WO2012103058A1 - Method for synthesizing glucans with beta-1,3 and beta-1,6 linkages - Google Patents

Abstract

The present invention provides methods for synthesizing beta-glucans, including beta (1,3) and beta (1,6) linkages, as well as intermediates and final products. Beta- glucans are important compounds for use in the development of anti-fungal therapies.

Description

METHOD FOR SYNTHESIZING GLUCANS WITH BET A- 1,3 AND

BET A- 1,6 LINKAGES

BACKGROUND

1. Technical Field text

[0001] This invention relates to synthetic glucans containing beta- 1,3 and beta- 1 ,6 linkages and intermediates thereof.

2. Background Information

[0002] Fungi are characterized by a cell wall rich in carbohydrates in the form of polysaccharides or glycoproteins. Among these, beta-glucans are the most represented in all fungi, conferring mechanical strength to the cell wall, and the associated osmotic resistance to intracellular pressure. Beta-glucans have been shown to contain one or more protective epitope(s) against C. albicans in mice. Moreover, the basic molecular structure of beta-glucan polymers is similar and highly conserved in different pathogenic fungal species, mainly consisting of beta- (1,3)- and beta-(l,6)-linked repeating units of d-glucose cross-linked together, and variously complexed with chitin and other glycoproteins (Masuoka, Clin

Microbiol Rev 2004;17:281-310).

[0003] Berti and colleagues have used various beta-glucan antigens to generate vaccines against fungal diseases. In a first attempt, they used laminarin from Laminaria digitata (Lam), a beta-glucan from a non- fungal source whose molecular structure resembles that of fungal beta-glucans, to generate an experimental glycoconjugate vaccine having the non-toxic mutant of diphtheria toxin CRM197 as the carrier. This conjugate (Lam-CRM197), formulated with Freund's adjuvant (CFA), was able to induce in mice specific anti beta-glucan antibodies that resulted protective against a lethal challenge with either pathogenic C. albicans and A. fumigatus (Torosantucci et al, JEM 2005; 202 (5):597-606) suggesting the possibility that a single vaccination could be used for protection against different fungal pathogens. This vaccine format had, however, some limitations. A first one was the use of CFA as the adjuvant. CFA is very potent and widely employed for preclinical investigations in animal models, but is not acceptable for human use, due to its high local toxicity. This precluded any possibility of testing the vaccine in a human setting. A second issue was the relative complexity of the beta-glucan antigen used in the conjugate. Lam is an heterogeneous, branched polysaccharide containing both beta-(l,3)- and beta- (l,6)-linked d-glucose sequences and, upon Lam-CRM197 vaccination, a composite array of antibodies, binding either beta-(l ,3)- or beta-(l,6)-glucans, was elicited in mice (Torosantucci et al., supra; Read et al., Carbohydr Res 1996; 281 : 187-201 ; Kim et al., Carbohydr Res 2000; 328:331-41). Thus, Berti and colleagues concluded that it was "difficult to discern, in Lam-CRM197 vaccine, the most protective beta-glucan epitope(s), as it would be desirable for further vaccine" (Bromuro et al., Vaccine 28(14):2615-23).

[0004] Berti et al. also described a series of different beta-glucan-based, conjugated vaccines, using linear, natural beta-(l ,3)-glucans or synthetic, linear or branched, beta-glucan oligosaccharides (See Figure 1 of Bromuro et al., supra). These conjugates were formulated with the human-acceptable adjuvant MF59, an oil-in- water emulsion and compared for their immunogenicity and protective performances against experimental infections with the fungal pathogen Candida albicans (Bromuro et al., supra; WO 09/077854 and WO 09/068996). They reported that all of the tested conjugates, adjuvanted with MF59, were

immunogenic and elicited antibodies against both Lam and the native beta-glucan from Candida (GGZym). Interestingly, however, the conjugates clearly differed in ability to elicit anti beta-(l,6)-glucan (pustulan) antibodies: the 17mer-CRMl 97 conjugate resembled Lam-CRM197, since it also induced appreciable levels of antibodies directed against beta-(l,6)-glucan. On the other hand, the 15-mer conjugates induced very low titers, if any, of these antibodies and, finally, Curd- CRM197 did not induce them at all. When tested in active protection

experiments, Curd-CRM197 and 15mer-CRM197, but not 17mer-CRM, proved able to confer significant protection to mice.

[0005] Based on this single result with the 17mer-CRM, Berti and colleagues suggest that the protective beta-glucan epitope of Lam-CRM 197 vaccine is a linear, beta-(l,3)-linked glucose sequence with no beta-(l,6)-linked branches and optimum DP of 8 glucose units (Bromuro et al, supra). Nonetheless, in their publications and patent filings, they recognize that beta-(l,6)-linked branches may still play an important role as protective antigens (See e.g., Torosantucci et al., PLoS ONE 2009, 4(4): e5392; WO 09/077854 and WO 09/068996).

[0006] Ensley (US 2006/0084630) provides a method for forming beta- glucoside linkages in carbohydrate polymers, particularly synthetic glucan molecules.

BRIEF SUMMARY

[0007] The present invention provides methods of synthesizing beta-glucans with beta- 1,3 and beta- 1,6 linkages. The present invention provides for the synthesis of a beta-glucan with a specific number of monosaccharide units (i.e. it is a single molecular entity; as opposed to, e.g., a composition containing a variety of beta-glucans with an average number of monosaccharide units).

[0008] In one embodiment, the present invention provides a method for forming a beta-glucan with a beta (1,6) bond comprising the step of (a) reacting for a time sufficient to form a beta (1,3) linkage (i) a compound (IV)

8 and (ii) a compound (III)

(III)

[0010] where al is 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10;

[0011] bl is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10; wherein Kal+bl < 15;

[0012] X is a bond, a linker or a protected linker;

[0013] Y is absent, -H, or a solid support.

[0014] In some embodiments, the intermediate (IV) is

[0015] In another embodiment, the present invention provides a method for forming a beta-glucan with a beta (1 ,3) bond comprising the step of (b) reacting for a time sufficient to form a beta (1 ,3) linkage (i) an intermediate (V)

[0016] where m is 1, 2, 3, 4, 5, 6, 7, or 8 and (ii) a compound (III)

(III)

[0017] where al is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;

[0018] bl is 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10; wherein Kal+bl < 15;

[0019] X is a bond, a linker or a protected linker;

[0020] Y is absent, -H, or a solid support.

[00211 In a preferred embodiment, the compound (V) is

[0022] The present invention also provides a synthetic beta-glucan of the formula (I):

[0023] where a is 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10;

[0024] b is 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10;

[0025] c is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10; wherein 2<a+b+c < 15;

[0026] d is 1 , 2, 3, 4, 5; wherein each d unit (a+b+c) can be the same or different;

[0027] X is a bond, a linker or a protected linker;

[0028] Y is absent, -H, a carrier or a solid support.

[0029] The present invention also provides a synthetic beta-glucan of the formula (II):

[0030] where a is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;

[0031] b is 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10;

[0032] c is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10; wherein 2<a+b+c < 15;

[0033] d is 1 , 2, 3, 4, 5; wherein each d unit (a+b+c) can be the same or different;

[0034] X is a bond or a linker;

[0035] Y is absent, -H, a solid support or a carrier,

[0036] provided that if b=0, X=a bond and Y =H, then a+b≠ 10 or 15 and further provided that if a=6, b=l , X=a bond and Y =H, then c≠ 2. [0037] The present invention also provides various intermediates useful for synthesizing beta-glucans, including compound 3, 4, 13, 14, 15, 16, 26, 27, 28, 29, 30, 31, 34, 35, 36, 37, 38, 41, 42, 43, 44, and 45 as shown in Figures 2-10.

BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1. Reproduction of Figure 1 from Bromuro et al, supra, showing the chemical structures of various tested beta-glucans. See also Figure 4 of WO 09/077854.

[0039] Figure 2. Monomer and dimer building blocks (3, 4, 5 and 6) useful for synthesis of beta-glucan targets (e.g., 1, 2).

[0040] Figure 3. Schematic of synthesis of building blocks 3 and 4.

[0041] Figure 4. Schematic of synthesis of various beta-glucans with beta (1,3) linkages.

[0042] Figure 5. Schematic of deprotection of a beta-glucan with beta (1 ,3) linkages.

[0043] Figure 6. Schematic of synthesis of building block 31.

[0044] Figure 7 Schematic of synthesis of various beta-glucans with beta (1,3) and beta (1,6) linkages.

[0045] Figure 8. Schematic of deprotection of a beta-glucan with beta ( 1 ,3) and beta (1 ,6) linkages.

[0046] Figure 9. Schematic of synthesis of building blocks 42 and 45.

[0047] Figure 10. Schematic of synthesis of building block 6.

DETAILED DESCRIPTION OF THE DRAWINGS AND THE PRESENTLY PREFERRED EMBODIMENTS

[0048] Definitions

[0049] In order to provide a clear and consistent understanding of the specification and claims, the following definitions are provided.

[0050] Units, prefixes, and symbols may be denoted in their SI accepted form.

Numeric ranges recited herein are inclusive of the numbers defining the range and include and are supportive of each integer within the defined range. Unless otherwise noted, the terms "a" or "an" are to be construed as meaning "at least one of." The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including but not limited to patents, patent applications, articles, books, and treatises, are hereby expressly incorporated by reference in their entirety for any purpose.

[0051] As used herein, "oligosaccharide" refers to a compound containing two or more monosaccharides. Oligosaccharides are considered to have a reducing end and a non-reducing end, whether or not the monosaccharide at the reducing end is in fact a reducing sugar. In accordance with accepted nomenclature, oligosaccharides are depicted herein with the non-reducing end on the left and the reducing end on the right. All oligosaccharides described herein are described with the name or abbreviation for the non-reducing monosaccharide (e.g., Glc), preceded by the configuration of the glycosidic bond (a or β), the ring bond, the ring position of the reducing monosaccharide involved in the bond, and then the name or abbreviation of the reducing monosaccharide (e.g., GlcNAc). The linkage between two sugars may be expressed, for example, as 2,3, 2→3, or 2-3. Each monosaccharide is a pyranose or furanose.

[0052] As used herein, "monosaccharide" refers to a single sugar residue in an oligosaccharide, including derivatives therefrom. Within the context of an oligosaccharide, an individual monomer unit is a monosaccharide which is (or can be) bound through a hydroxyl group to another monosaccharide.

[0053] As used herein, "endotoxin-free" refers to an oligosaccharide that does not contain endotoxins or endotoxin components normally present in bacterial isolates.

[0054] As used herein, "synthetic" refers to material which is substantially or essentially free from components, such as endotoxins, glycolipids,

oligosaccharides, etc., which normally accompany a compound when it is isolated. Typically, synthetic compounds are at least about 90% pure, usually at least about 95%, and preferably at least about 99% pure. Purity can be indicated by a number of means well known in the art. Preferably, purity is measured by HPLC. The identity of the synthetic material can be determined by mass spectroscopy and/or NMR spectroscopy.

[0055] As used herein, the term "carrier" refers to a protein, peptide, lipid, polymer, dendrimer, virosome, virus-like particle (VLP), or combination thereof, which is coupled to the oligosaccharide to enhance the immunogenicity of the resulting oligosaccharide-carrier conjugate to a greater degree than the

oligosaccharide alone.

[0056J As used herein, "protein carrier" refers to a protein, peptide or fragment thereof, which is coupled or conjugated to an oligosaccharide to enhance the immunogenicity of the resulting oligosaccharide-protein carrier conjugate to a greater degree than the oligosaccharide alone. For example, when used as a carrier, the protein carrier may serve as a T-dependent antigen which can activate and recruit T-cells and thereby augment T-cell dependent antibody production.

[0057] As used herein, "conjugated" refers to a chemical linkage, either covalent or non-covalent, that proximally associates an oligosaccharide with a earner so that the oligosaccharide conjugate has increased immunogenicity relative to an unconjugated oligosaccharide.

[0058] As used herein, "conjugate" refers to an oligosaccharide chemically coupled to a earner through a linker and/or a cross-linking agent.

[0059] As used herein, "passive immunity" refers to the administration of antibodies to a subject, whereby the antibodies are produced in a different subject (including subjects of the same and different species) such that the antibodies attach to the surface of the bacteria and cause the bacteria to be phagocytosed or killed.

[0060] As used herein, "protective immunity" means that a vaccine or immunization schedule that is administered to a animal induces an immune response that prevents, retards the development of, or reduces the severity of a disease that is caused by a pathogen or diminishes or altogether eliminates the symptoms of the disease. Protective immunity may be predicted based on the ability of serum antibody to activate complement-mediated bactericidal activity or confer passive protection against a bacterial infection in a suitable animal challenge model. [0061] As used herein, "immunoprotective composition" refers to a

composition formulated to provide protective immunity in a host.

[0062] As used herein, "in a sufficient amount to elicit an immune response" (e.g., to epitopes present in a preparation) means that there is a detectable difference between an immune response indicator measured before and after administration of a particular antigen preparation. Immune response indicators include but are not limited to: antibody titer or specificity, as detected by an assay such as enzyme-linked immunoassay (ELISA), bactericidal assay (e.g., to detect serum bactericidal antibodies), flow cytometry, immunoprecipitation, Ouchter- Lowry immunodiffusion; binding detection assays of, for example, spot, Western blot or antigen arrays; cytotoxicity assays, and the like.

[0063] As used herein, "antibody" encompasses polyclonal and monoclonal antibody preparations, as well as preparations including hybrid antibodies, altered antibodies, F(ab')² fragments, F(ab) molecules, Fv fragments, single chain fragment variable displayed on phage (scFv), single domain antibodies, chimeric antibodies, humanized antibodies, and functional fragments thereof which exhibit immunological binding properties of the parent antibody molecule.

[0064] As used herein, "monoclonal antibody" refers to an antibody composition having a homogeneous antibody population. The term is not limited by the manner in which it is made. The term encompasses whole immunoglobulin molecules, as well as Fab molecules, F(ab')₂ fragments, Fv fragments, single chain fragment variable displayed on phage (scFv), and other molecules that exhibit immunological binding properties of the parent monoclonal antibody molecule.

[0065] As used herein, "specifically binds to an antibody" or "specifically immunoreactive with", when referring to an oligosaccharide, protein or peptide, refers to a binding reaction which is based on and/or is probative of the presence of the antigen in a sample which may also include a heterogeneous population of other molecules. Thus, under designated immunoassay conditions, the specified antibody or antibodies bind(s) to a particular antigen or antigens in a sample and does not bind in a significant amount to other molecules present in the sample.

Specific binding to an antibody under such conditions may require an antibody or antiserum that is selected for its specificity for a particular antigen or antigens. [0066] As used herein, "antigen" refers to include any substance that may be specifically bound by an antibody molecule.

[0067] As used herein, "immunogen" and "immunogenic composition" refer to an antigenic composition capable of initiating lymphocyte activation resulting in an antigen-specific immune response.

[0068] As used herein, "epitope" refers to a site on an antigen to which specific B cells and/or T cells respond. The term is also used interchangeably with

"antigenic determinant" or "antigenic determinant site." B cell epitope sites on proteins, oligosaccharides, or other biopolymers may be composed of moieties from different parts of the macromolecule that have been brought together by folding. Epitopes of this kind are referred to as conformational or discontinuous epitopes, since the site is composed of segments the polymer that are

discontinuous in the linear sequence but are continuous in the folded

conformation(s). Epitopes that are composed of single segments of biopolymers or other molecules are termed continuous or linear epitopes. T cell epitopes are generally restricted to linear peptides. Antibodies that recognize the same epitope can be identified in a simple immunoassay showing the ability of one antibody to block the binding of another antibody to a target antigen. [0069] Synthetic oligosaccharides

[0070] The present invention provides synthetic beta glucans of the formula (I) or (II) as set forth above.

[0071] Beta-glucans in accordance with the present invention can be formed from the building blocks 3, 4, 5 and 6 as shown in Figures 2-10. Compound 10 was previously described by Izumi et al., Biosci. Biotechnol. Biochem., 2002, 66(1):211 and Yang et al., Carbohydrate Research, 2000, 329:879-884.

[0072] Suitable linkers (X) comprise at one end a functional group able to enter into a covalent bonding with a reactive functional group of a carrier, e.g. an amino, thiol, or carboxyl group, and at the other end a functional group likewise able to enter into a covalent bonding with a hydroxyl group of the beta-glucan according to the present invention. Between the two functional groups of the linker molecule there is a biocompatible bridging molecule of suitable length, e.g. substituted or unsubstituted heteroalkylene, arylalkylene, alkylene, alkenylene, or (oligo)alkylene glycol groups. Linkers preferably include substituted or unsubstituted alkylene or alkenylene groups containing 1-10 carbon atoms.

[0073] Functional groups able to react with thiol groups on the carrier are, for example, maleimide and carboxyl groups; preferred functional groups able to react with aldehyde or carboxyl groups on the carrier are, for example, amino or thiol groups. Preferred covalent attachments between linkers and carriers include thioethers from reaction of a thiol with an a-halo carbonyl or a-halo nitrile, including reactions of thiols with maleimide; hydrazides from reaction of a hydrazide or hydrazine with an activated carbonyl group (e.g. activated NHS-ester or acid halide); triazoles from reaction of an azide with an alkyne (e.g. via "click chemistry"); and oximes from reaction of a hydroxylamine and an aldehyde or ketone as disclosed, for example, in Lees et al., Vaccine, 24:716, 2006. Although amine-based conjugation chemistries could be used in principle for coupling linkers and/or spacers to the oligosaccharides described herein, these approaches would typically sacrifice uniformity inasmuch as the oligosaccharides of the present invention typically contain a plurality of amines bonded to second carbon of the respective monosaccharide units.

[0074] Further suitable linker molecules are known to skilled workers and commercially available or can be designed as required and depending on the functional groups present and can be prepared by known methods.

[0075] As used herein, a "protected linker" is a linker in which the functional group that is capable of binding to the carrier or solid support is protected with a suitable protecting group. Suitable carboxyl, hydroxyl and amino protecting groups are those customarily considered in carbohydrate chemistry, including those mentioned in "Protective Groups in Organic Synthesis", 3^ld edition, T. W. Greene and P. G. M. Wuts (Ed.), John Wiley and Sons, New York, 1999. See also ocienski, Philip J.; Protecting Groups (Georg Thieme Verlag Stuttgart, New York, 1994), which is incorporated by reference in its entirety (in particular Chapter 1, Protecting Groups: An Overview, pages 1-20, Chapter 2, Hydroxyl

Protecting Groups, pages 21-94, Chapter 3, Diol Protecting Groups, pages 95-117, Chapter 4, Carboxyl Protecting Groups, pages 118-154, Chapter 5, Carbonyl Protecting Groups, pages 155-184).

[0076] Exemplary amino-protecting groups (for protecting an amino functional group on the linker which binds to the carrier/solid support) are azide groups, silyl groups such as trimethylsilyl, triisopropylsilyl, tributylsilyl, t-butyldimethylsilyl and t-butyldiarylsilyl; carbamates such as (trichloroethyl) carbamate (Troc), t- butoxycarbonyl, benzyloxycarbonyl, 4-methoxybenzyloxycarbonyl and 4- nitrobenzyloxycarbonyl; formyl, acetyl (Ac), benzoyl and pivaloyl,

methoxymethyl, t-butyl, benzyl and tetrahydropyranyl. A preferred amino- protecting group is azido, benzyl, and benzyloxycarbonyl, most preferably azido.

[0077] Typical hydroxy protecting groups (for protecting either the hydroxyl group of the reducing terminus of the forming beta-glucan or a hydroxyl functional group of the linker which binds to the carrier/solid support) include, for example, substituted methyl and alkyl ethers, substituted benzyl ethers, silyl ethers, esters including sulfonic acid esters, and carbonates. Specific examples include pivaloyl, benzylidene, formyl, acetyl, substituted acetyl, propionyl, butynyl, pivalamido, benzoyl, biphenylcarbonyl, substituted biphenylcarbonyl, ethoxycarbonyl, t-butoxycarbonyl, benzyloxycarbonyl, phenoxycarbonyl, benzyl, diphenylmethyl, triphenylmethyl, t-butyl, tetrahydropyranyl, alkylenes such as allyl (All) and allyl derivatives, N-phenylcarbamate, N-imidazoyl carbamate, trialkylsilyl, isopropyldialkylsilyl, alkyldiisopropylsilyl, triisopropylsilyl and t- butyldialkylsilyl. The preferred group for protecting the hydroxyl group of the reducing terminus of the forming beta-glucan are alkylenes, more preferable allyl or derivatives thereof.

[0078] Examples of protecting groups for carboxyl groups include, for example, benzyl ester, cyclohexyl ester, 4-nitrobenzyl ester, t-butyl ester, 4- pyridylmethyl ester, and the like.

[0079] Suitable carriers are known in the art (See e.g., Remington's

Pharmaceutical Sciences (18th ed., Mack Easton, PA (1990)) and may include, for example, proteins, peptides, lipids, polymers, dendrimers, virosomes, virus-like particles (VLPs), or combinations thereof, which by themselves may not display particular antigenic properties, but can support immunogenic reaction of a host to the oligosaccharides of the present invention (antigens) displayed at the surface of the carrier(s).

[0080] Preferably, the carrier is a protein carrier, including but are not limited to, bacterial toxoids, toxins, exotoxins, and nontoxic derivatives thereof, such as tetanus toxoid, tetanus toxin Fragment C, diphtheria toxoid, CRM (a nontoxic diphtheria toxin mutant) such as CRM 197, cholera toxoid, Staphylococcus aureus exotoxins or toxoids, Escherichia coli heat labile enterotoxin, Pseudomonas aeruginosa exotoxin A, including recombinantly produced, genetically detoxified variants thereof; bacterial outer membrane proteins, such as Neisseria meningitidis serotype B outer membrane protein complex (OMPC), outer membrane class 3 porin (rPorB) and other porins; keyhole limpet hemocyanine (KLH), hepatitis B virus core protein, thyroglobulin, albumins, such as bovine serum albumin (BSA), human serum albumin (HSA), and ovalbumin; pneumococcal surface protein A (PspA), pneumococcal adhesin protein (PsaA); purified protein derivative of tuberculin (PPD); transferrin binding proteins, polyamino acids, such as

poly(lysine:glutamic acid); peptidyl agonists of TLR-5 (e.g. flagellin of motile bacteria like Listeria); and derivatives and/or combinations of the above carriers. Preferred carriers for use in humans include tetanus toxoid, CRM 197, and OMPC.

[0081] Depending on the type of bonding between the linker and the carrier, and the structural nature of the carrier and oligosaccharide, a carrier may display on average, for example, 1 to 500, 1 to 100, 1 to 20, or 3 to 9 oligosaccharide units on its surface.

[0082] Methods for attaching an oligosaccharide to a carrier, such as a carrier protein are conventional, and a skilled practitioner can create conjugates in accordance with the present invention using conventional methods. Guidance is also available in various disclosures, including, for example, U.S. Pat. Nos.

4,356,170; 4,619,828; 5,153,312; 5,422,427; and 5,445,817; and in various print and online Pierce protein cross-linking guides and catalogs (Thermo Fisher, Rockford, IL).

[0083] In one embodiment, the carbohydrate antigens of the present invention are conjugated to CRM 197, a commercially available protein carrier used in a number of FDA approved vaccines. CRM- conjugates have the advantage of being easier to synthesize, purify and characterize than other FDA approved carriers such as OMPC. Carohydrate antigens may be conjugated to CRM via thiol- bromoacetyl conjugation chemistry. CRM activation may be achieved by reacting the lysine side chains with the NHS ester of bromoacetic acid using standard conditions as previously described in U.S. Pat. Appl. Publ. 2007-

0134762, the disclosures of which are incorporated by reference herein. CRM may be functionalized with 10-20 bromoacetyl groups per protein (n= 10-20) prior to conjugation. Conjugation may be performed at pH=9 to avoid aggregation of CRM. Careful monitoring of pH must be employed to ensure complete CRM reaction with NHS-bromoacetate while minimizing background hydrolysis of

CRM. Activated CRM may be purified by size exclusion chromatography prior to conjugation. Antigen-CRM conjugates may be synthesized by reacting thiol- terminated carbohydrate antigens with bromoacetamide-activated CRM.

[0084] CRM conjugates may be purified via size exclusion chromatography to remove and recover any unreacted carbohydrate. MBTH (specific for GlcNAc residues) and Bradford assays may be used to determine carbohydrate:protein ratio and protein content, respectively, as previously described (Manzi et al., Curr. Prot. Mol. Biol, section 17.9.1 (Suppl. 32), 1995. In preferred embodiments, a minimum carbohydrate content of about 15% by weight for each conjugate may be generated. Typically, a conjugate may include about 3-20 antigens per protein carrier.

[0085] Suitable solid supports to which the beta-glucan can be covalently attached include physical solids as well as insoluble polymers, insoluble particles, surfaces, membranes and resins, preferably the solid support is an insoluble polymer, an insoluble particle, a surface or a resin. Thus the "solid support" may be an insoluble inorganic matrix (such as glass), an insoluble polymer (such as a plastic, for example polystyrene), an insoluble matrix consisting of parts of both organic and inorganic components (e.g. some hybrid silicates, such as compounds of the structure R-Si-O-), organic polymers in common use in solid-phase synthesis (polystyrenes, PEGA resins, PEG resins, SPOCC resins and hybrids thereof), polyethylene glycol chains (which can be soluble in certain organic solvents and made insoluble by the addition of other solvents). The solid may also be a metal (such as gold), an alloy, or a composite such as for example indium-tin oxide or mica.

[0086] Any of the above listed solid supports may additionally be coated with agents that have an affinity for carbohydrates, such as but not limited to aryl boronates or polymers thereof. Such coatings can increase the concentration of carbohydrate at the surface of the solid support, enhancing the rate and yield of capture.

[0087] Organic polymers used in solid-phase synthesis for example includes TentaGel (commercially available from Rapp polymere, Tubingen, Germany), ArgoGel (commercially available from Argonaut Technologies Inc., San Carlos, Calif), PEGA (commercially available from Polymer Laboratories, Amherst, Mass.), POEPOP (Renil et al., 1996, Tetrahedron Lett., 37: 6185-88; available from Versamatrix, Copenhagen, Denmark) and SPOCC (Rademann et al, 1999, J. Am. Chem. Soc, 121 : 5459-66; available from Versamatrix, Copenhagen, Denmark).

[0088] In one embodiment of the invention the solid support is a sensor, such as a surface acoustic wave sensor (such as any of the sensors described in

Samoyolov et al. 2002, J. Molec. Recognit. 15: 197-203) or a surface plasmon resonance sensor (such as any of the sensors reviewed by Homola et al., 1999, Sensors and Actuators B, 54: 3-15). Such solid supports may be inorganic materials such as glass, metals such as gold, organic polymeric materials or hybrids thereof and may be covered various coatings such as proteins or polysaccharides, oligomers such as dendrimers or polymers such as

polyacryl amide or polyethylene glycol. In a preferred embodiment the solid support is glass or PEGA resins.

[0089] In one embodiment of the present invention the solid support is coupled to a reference standard, which may facilitate quantification of immobilised reducing sugar. In particular, it is preferred that said reference standard is attached to the solid support (either directly or indirectly) by a cleavable linker, which could facilitate quantification of immobilised and released reducing sugar. In embodiments of the invention wherein the reducing sugar is immobilised to the solid support via a cleavable linker, it is preferred that the reference standard is immobilised to the solid support via an identical or similar cleavable linker.

[0090] The reference standard may be any detectable compound, for example the reference standard may or may not be a sugar, preferably however it is carbohydrate.

[0091] The amount of reference standard may vary, in general the solid support may comprise in the range of 20 to 500, preferably in the range of 50 to 200, such as in the range of 90 to 1 10-NH₂ groups per reference standard.

[0092] In another embodiment, beta-glucan antigens may be conjugated to one or more carriers/solid supports suitable for development of diagnostic assays, including ELISAs and microarrays. Exemplary carriers for use in such assays include bovine serum albumin (BSA), keyhole limpet hemocyanine (KLH), biotin, a label, a glass slide or a gold surface. By way of example, synthetic carbohydrate antigens may be conjugated to BSA by a thiol-maleimide coupling procedure. Maleimide-BSA contains 15-20 maleimide groups per protein (n=l 5-20).

Accordingly, oligosaccharide antigens may be conjugated to maleimide

functionalized BSA, whereby a 20-fold molar excess of the antigen is reacted with commercially available Imject maleimide BSA (Pierce) in maleimide conjugation buffer (Pierce). Conjugation may be performed at pH=7.2 to avoid hydrolysis of the maleimide group during conjugation.

[0100] In another aspect, the present invention provides compositions containing beta-glucans (II) and a pharmaceutically acceptable vehicle. The compositions are preferably immunogenic and immunoprotective.

[0101] The present invention contemplates the use of single- and multi-valent vaccines comprising any of the synthetic oligosaccharides described herein. The identification of a single oligosaccharide antigen eliciting a protective immune response can facilitate development of a single-antigen vaccine candidate against one or more bacterial target(s) expressing a beta-glucan (II). Thus, in one embodiment, the compositions may contain a single beta-glucan (II).

[0102| The present invention further contemplates multi-antigen vaccine candidates and vaccines thereof. In one embodiment, the invention provides a composition containing two, three, four or more different beta-glucans (II). [0103] Suitable vehicles and their formulation, inclusive of other human proteins, e.g., human serum albumin, are described, for example, in Remington's Pharmaceutical Sciences (18th ed., Mack Easton Pa. (1990)). Pharmaceutically acceptable vehicles may include any vehicle that does not itself induce the production of antibodies harmful to the individual receiving the composition. Suitable vehicles may include large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers; inactive virus particles, insoluble aluminum compounds, calcium phosphate, liposomes, virosomes, ISCOMS, microparticles, emulsions, and VLPs.

[0104] The compositions of the present invention may further include one or more adjuvants. An oligosaccharide-protein conjugate composition may further include one or more immunogenic adjuvant(s). An immunogenic adjuvant is a compound that, when combined with an antigen, increases the immune response to the antigen as compared to the response induced by the antigen alone so that less antigen can be used to achieve a similar response. For example, an adjuvant may augment humoral immune responses, cell-mediated immune responses, or both.

[0105] Those of skill in the art will appreciate that the terms "adjuvant," and "carrier," can overlap to a significant extent. For example, a substance which acts as an "adjuvant" may also be a "carrier," and certain other substances normally thought of as "earners," for example, may also function as an "adjuvant."

Accordingly, a substance which may increase the immunogenicity of the synthetic oligosaccharide or carrier associated therewith is a potential adjuvant. As used herein, a carrier is generally used in the context of a more directed site-specific conjugation to an oligosaccharide of the present invention, whereby an adjuvant is generally used in a less specific or more generalized structural association therewith.

[0106] Exemplary adjuvants and/or adjuvant combinations may be selected from the group consisting of mineral salts, including aluminum salts, such as aluminum phosphate and aluminum hydroxide (alum) (e.g., Alhydrogel™,

Superfos, Denmark) and calcium phosphate; PJBI, which contains three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate, and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween 80 emulsion, whereby any of the 3 components MPL, TDM or CWS may also be used alone or combined 2 by 2; toll-like receptor (TLR) agonists, including, for example, agonists of TLR- 1 (e.g. tri-acyl lipopeptides); agonists of TLR- 2 (e.g. peptidoglycan of gram-positive bacteria like streptococci and staphylococci;

lipoteichoic acid); agonists of TLR-3 (e.g. double-stranded RNA and their analogs such as poly 1 :C); agonists of TLR-4 (e.g. lipopolysaccharide (endotoxin) of gram-negative bacteria like Salmonella and E. coli); agonists of TLR- 5 (e.g.

flagellin of motile bacteria like Listeria); agonists of TLR-6 (e.g. with TLR-2 peptidoglycan and certain lipids (diacyl lipopeptides)); agonists of TLR- 7 (e.g. single- stranded RNA (ssRNA) genomes of such viruses as influenza, measles, and mumps; and small synthetic guanosine-base antiviral molecules like loxoribine and ssRNA and their analogs); agonists of TLR-8 (e.g. binds ssRNA); agonists of TLR-9 (e.g. unmethylated CpG of the DNA of the pathogen and their analogs; agonists of TLR- 10 (function not defined) and TLR- 11 -(e.g. binds proteins expressed by several infectious protozoans (Apicomplexa), specific toll-like receptor agonists include monophosphoryl lipid A (MPL®), 3 De-O-acylated monophosphoryl lipid A (3 D-MPL), OM-174 (E. coli lipid A derivative); OM triacyl lipid A derivative, and other MPL- or lipid A-based formulations and combinations thereof, including MPL®-SE, RC-529 (Dynavax Technologies), AS01 (liposomes+MPL+QS21), AS02 (oil-in- water PL + QS-21), and AS04 (Alum + MPL)(GlaxoSmith Kline, Pa.), CpG-oligodeoxynucleotides (ODNs) containing immunostimulatory CpG motifs, double-stranded RNA,

polyinosinic:polycytidylic acid (poly I:C), and other oligonucleotides or polynucleotides optionally encapsulated in liposomes; oil-in-water emulsions, including AS03 (GlaxoSmith Kline, Pa.), MF-59 (microfluidized detergent stabilized squalene oil-in-water emulsion; Novartis), and Montanide IS A-51 VG (stabilized water-in-oil emulsion) and Montanide ISA-720 (stabilized

water/squalene; Seppic Pharmaceuticals, Fairfield, NJ); cholera toxin B subunit; saponins, such as Quil A or QS21, an HPLC purified non-toxic fraction derived from the bark of Quillaja Saponaria Molina (STIMULON™ (Antigenics, Inc.,

Lexington, Mass.) and saponin-based adjuvants, including immunostimulating complexes (ISCOMs; structured complex of saponins and lipids) and other ISCOM-based adjuvants, such as ISCOMATRIX™ and AbISCO^®-100 and -300 series adjuvants (Isconova AB, Uppsala, Sweden); QS21 and 3 D-MPL together with an oil in water emulsion as disclosed in U.S. Pat. Appl. No. 2006/0073171 ; stearyl tyrosine (ST) and amide analogs thereof; virus-like particles (VLPs) and reconstituted influenza virosomes (IRIVs); complete Freund's adjuvant (CFA); incomplete Freund's adjuvant (IF A); E. coli heat-labile enterotoxin (LT); immune- adjuvants, including cytokines, such as IL-2, IL-12, GM-CSF, Flt3, accessory molecules, such as B7.1, and mast cell (MC) activators, such as mast cell activator compound 48/80 (C48/80); water-insoluble inorganic salts; liposomes, including those made from DNPC/Chol and DC Choi; micelles; squalene; squalane;

muramyl dipeptides, such as N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr- MDP) as found in U.S. Pat. No. 4,606,918, N-acetyl-normuramyl-L-alanyl-D- isoglutamine (nor-MDP), and N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L- alanine-2-(r2'-dipalmitoyl-n-glycero-3-hydroxyphosphoryl; SAF-1 (Syntex); AS05 (GlaxoSmith Kline, Pa.); and combinations thereof.

[0107] In preferred embodiments, adjuvant potency may be enhanced by combining multiple adjuvants as described above, including combining various delivery systems with immunopotentiating substances to form multi-component adjuvants with the potential to act synergistically to enhance antigen-specific immune responses in vivo. Exemplary immunopotentiating substances include the above-described adjuvants, including, for example, MPL and synthetic derivatives, MDP and derivatives, oligonucleotides (CpG etc), ds RNAs, alternative pathogen- associated molecular patterns (PAMPs)(E. coli heat labile enterotoxin; flagellin, saponins (QS-21 etc), small molecule immune potentiators (SMIPs, e.g., resiquimod (R848)), cytokines, and chemokines.

[0108] Methods of Treating or Preventing Fungal infections

[0109] Anti-fungal compositions

[0110] In one embodiment, the present invention provides pharmaceutically acceptable immunogenic or immunoprotective beta-glucan (II) compositions and their use in methods for preventing a fungal infection in a patient in need thereof. In one embodiment, comprising administering an effective amount of an oligosaccharide of the present invention. An immunogenic or immunoprotective composition will include a "sufficient amount" or "an immunologically effective amount" of a beta-glucan conjugate according to the present invention, as well as any of the above mentioned components, for purposes of generating an immune response or providing protective immunity, as further defined herein.

[0111] Administration of the beta-glucan conjugate compositions or antibodies, as described herein may be carried out by any suitable means, including by parenteral administration (e.g., intravenously, subcutaneously, intradermally, or intramuscularly); by topical administration, of for example, antibodies to an airway surface; by oral administration; by in ovo injection in birds, for example, and the like. Preferably, they are administered

intramuscularly.

[0112] Typically, the compositions may be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection. An aqueous composition for parenteral administration, for example, may include a solution of the immunogenic component(s) dissolved or suspended in a pharmaceutically acceptable vehicle or diluent, preferably a primarily aqueous vehicle. An aqueous composition may be formulated as a sterile, pyrogen- free buffered saline or phosphate-containing solution, which may include a preservative or may be preservative free. Suitable preservatives include benzyl alcohol, parabens, thimerosal, chlorobutanol, and benzalkonium chloride, for example. Aqueous solutions are preferably

approximately isotonic, and its tonicity may be adjusted with agents such as sodium tartrate, sodium chloride, propylene glycol, and sodium phosphate.

Additionally, auxiliary substances required to approximate physiological conditions, including pH adjusting and buffering agents, tonicity adjusting agents, wetting or emulsifying agents, pH buffering substances, and the like, including sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc. may be included with the vehicles described herein. [0113] Compositions may be formulated in a solid or liquid form for oral delivery. For solid compositions, nontoxic and/or pharmaceutically acceptable solid vehicles may include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like. For oral administration, a

pharmaceutically acceptable nontoxic composition may be formed by

incorporating any of the normally employed excipients, including those vehicles previously listed, and a unit dosage of an active ingredient, that is, one or more compounds of the invention, whether conjugated to a carrier or not. Topical application of antibodies to an airway surface can be carried out by intranasal administration (e.g., by use of dropper, swab, or inhaler which deposits a pharmaceutical formulation intranasally). Topical application of the antibodies to an airway surface can also be carried out by inhalation administration, such as by creating respirable particles of a pharmaceutical formulation (including both solid particles and liquid particles) containing the antibodies as an aerosol suspension, and then causing the subject to inhale the respirable particles. Methods and apparatuses for administering respirable particles of pharmaceutical formulations are well known, and any conventional technique can be employed. Oral administration may be in the form of an ingestable liquid or solid formulation.

[0114] The preparation of such pharmaceutical compositions is within the ordinary skill in the art, and may be guided by standard reference books such as Remington the Science and Practice of Pharmacy, Lippincott Williams & Wilkins; 21 ed., May 1 , 2005, which is incorporated herein by reference.

[0115] The concentration of the oligosaccharides of the invention in the pharmaceutical formulations can vary widely, i.e., from less than about 0.1%, usually at or at least about 0.1% to as much as 20% to 50% or more by weight, and may be selected on the basis of fluid volumes, viscosities, stability, etc., and/or in accordance with the particular mode of administration selected. A human unit dose form of the compounds and composition is typically included in a

pharmaceutical composition that comprises a human unit dose of an acceptable vehicle, preferably an aqueous vehicle, and is administered in a volume of fluid that is known by those of skill in the art to be used for administration of such compositions to humans, and is adjusted according to commonly understood principles for a particular subject to be treated. Thus in one embodiment, the invention provides a unit dosage of the vaccine components of the invention in a suitable amount of an aqueous solution, such as 0.1-3 ml, preferably 0.2-2 mL.

[0116] The compositions of the present invention may be administered to any animal species at risk for developing an infection by a fungal species expressing a beta-glucan antigen.

[0117] The present invention can also be used to treat or prevent infections where the fungi or bacterium is known or suspected to express a beta-glucan (II), including Candida species, such as Calbicans; Cryptococcus species, such as Cneoformans; Enterococcus species, such as Efaecalis; Streptococcus species, such as Spneumoniae, Smutans, Sagalactiae and Spyogenes; Leishmania species, such as L. major; Acanthamoeba species, such as A.castellani; Aspergillus species, such as Afumigatus and A.flavus; Pneumocystis species, such as P.carinii;

Mycobacterium species, such as Mtuberculosis; Pseudomonas species, such as P. aeruginosa; Staphylococcus species, such as Saureus; Salmonella species, such as Styphimurium; Coccidioides species such as C immitis; Trichophyton species such as T verrucosum; Blastomyces species such as B.dermatidis; Histoplasma species such as Hcapsulatum; Paracoccidioides species such as P.brasiliensis; Pythium species such as P.insidiosum; and Escherichia species, such as E.coli.

[0118] The treatment may be given in a single dose schedule, or preferably a multiple dose schedule in which a primary course of treatment may be with 1-10 separate doses, followed by other doses given at subsequent time intervals required to maintain and or reinforce the response, for example, at 1 -4 months for a second dose, and if needed, a subsequent dose(s) after several months. Examples of suitable treatment schedules include: (i) 0, 1 month and 6 months, (ii) 0, 7 days and 1 month, (iii) 0 and 1 month, (iv) 0 and 6 months, or other schedules sufficient to elicit the desired responses expected to reduce disease symptoms, or reduce severity of disease.

[0119] The amounts effective for inducing an immune response or providing protective immunity will depend on a variety of factors, including the

oligosaccharide composition, conjugation to a carrier, inclusion and nature of adjuvant(s), the manner of administration, the weight and general state of health of the patient, and the judgment of the prescribing physician. By way of example, the amounts may generally range for the initial immunization (that is for a prophylactic administration) from about 1.0 μg to about 5,000 μg of

oligosaccharide for a 70 kg patient, (e.g., 1.0 μg, 2.0 μg, 2.5 μg, 3.0 μg, 3.5 μg, 4.0 μg, 4.5 μg, 5.0 μg, 7.5 μg, 10 g, 12.5 μ^ 15 μg, 17.5 μg, 20 μ¾ 25 μg, 30 μg, 35 μg, 40 μg, 45 μg, 50 μg, 75 μg, 100 μg, 250 μg, 500 μ^ 750 μg, 1 ,000 μg, 1 ,500 μ^ 2,000 μg, 2,500 μg, 3,000 μg, 3,500 μg, 4,000 μg, 4,500 μg or 5,000 μg). The actual dose administered to a subject is often, but not necessarily, determined according to an appropriate amount per kg of the subject's body weight. For example, an effective amount may be about 0.1 μg to 5 μg/kg body weight.

[0120] A primary dose may optionally be followed by boosting dosages of from about 1.0 to about 1 ,000 of peptide (e.g., 1.0 μg, 2.0 μg, 2.5 μg, 3.0 μg, 3.5 μg, 4.0 μg, 4.5 μ^ 5.0 μg, 7.5 μg, 10 μg, 12.5 μg, 15 μg, 17.5 μg, 20 μg, 25 μg, 30 μ_§, 35 μg, 40 μ_§, 45 μg, 50 μg, 75 μg, 100 μg, 250 μ_§, 500 μ_Ε, 750 μ_§, 1,000 μg, 1 ,500 μg, 2,000 μg, 2,500 μg, 3,000 μg, 3,500 μg, 4,000 μg, 4,500 μg or 5,000 μg) pursuant to a boosting regimen over weeks to months depending upon the patient's response and condition by measuring specific T cell activity in the patient's blood.

[0121] The immunogenic compositions comprising a compound of the invention may be suitable for use in adult humans or in children, including young children or others at risk for contracting an infection caused by a beta- 1 ,3 and beta 1 ,6 glucan-expressing bacterial species. Optionally such a composition may be administered in combination with other pharmaceutically active substances, and frequently it will be administered in combination with other vaccines as part of a childhood vaccination program.

[0122] Antibody Compositions

[0123] In another embodiment, the invention provides an antibody preparation against one or more oligosaccharides I in accordance with the present invention. The antibody preparation may include any member from the group consisting of polyclonal antibody, monoclonal antibody, mouse monoclonal IgG antibody, humanized antibody, chimeric antibody, fragment thereof, or

combination thereof.

[0124] Pharmaceutical antibody compositions may be used in a method for providing passive immunity against a bacterial target species of interest, including Candida and other *** -expressing bacteria. A pharmaceutical antibody composition may be administered to an animal subject, preferably a human, in an amount sufficient to prevent or attenuate the severity, extent of duration of the infection by the bacterial target species of interest.

[0125] The administration of the antibody may be either prophylactic (prior to anticipated exposure to a bacterial infection) or therapeutic (after the initiation of the infection, at or shortly after the onset of the symptoms). The dosage of the antibodies will vary depending upon factors as the subject's age, weight and species. In general, the dosage of the antibody may be in a range from about 1-10 mg/kg body weight. In a preferred embodiment, the antibody is a humanized antibody of the IgG or the IgA class. The route of administration of the antibody may be oral or systemic, for example, subcutaneous, intramuscular or intravenous.

[0126] Antibodies in diagnostic assays

[0127] In a further aspect, the present invention provides compositions and methods for inducing production of antibodies for diagnosing, treating, and/or preventing one or more infections caused by beta- glucan-expressing fungi or bacteria.

[0128] Antisera to glucan conjugates may be generated in New Zealand white rabbits by 3-4 subcutaneous injections over 13 weeks. A pre-immune bleed may generate about 5 mL of baseline serum from each rabbit. A prime injection (10 μg antigen equivalent) may be administered as an emulsion in complete

Freund' s adjuvant (CFA). Subsequent injections (5 μg antigen equivalent) may be given at three week intervals in incomplete Freund' s adjuvant (IF A). Rabbits may be bled every two weeks commencing one week after the third immunization. Approximately 25 - 30 mL of serum per rabbit may be generated from each bleeding event and frozen at -80°C. Serum may be analyzed by ELISA against the corresponding beta-glucan (II) conjugate as described below. In addition, antisera from later bleeds may be affinity purified as further described below.

[0129] The oligosaccharides and antibodies generated therefrom can be used as diagnostic reagents for detecting beta-glucan (II) structures or antibodies thereagainst, which are present in biological samples. The detection reagents may be used in a variety of immunodiagnostic techniques, known to those of skill in the art, including ELISA- and microarray-related technologies. In addition, these reagents may be used to evaluate antibody responses, including serum antibody levels, to immunogenic oligosaccharide conjugates. The assay methodologies of the invention typically involve the use of labels such as fluorescent,

chemiluminescent, radioactive, enzymatic labels or dye molecules, and/ or secondary immunologic reagents for direct or indirect detection of a complex between an antigen or antibody in a biological sample and a corresponding antibody or antigen bound to a solid support.

[0130] Such assays typically involve separation of unbound antibody in a liquid phase from a solid phase support to which antibody-antigen complexes are bound. Solid supports which can be used in the practice of the invention include substrates such as nitrocellulose (e.g., in membrane or microtiter well form);

polyvinylchloride (e.g., sheets or microtiter wells); polystyrene latex (e.g., beads or microtiter plates); polyvinylidine fluoride; diazotized paper; nylon membranes; activated beads, magnetically responsive beads, and the like.

[0131] Typically, a solid support is first reacted with a first binding component (e.g., an anti-beta-glucan (II) antibody or beta-glucan (II)) under suitable binding conditions such that the first binding component is sufficiently immobilized to the support. In some cases, mobilization to the support can be enhanced by first coupling the antibody or oligosaccharide to a protein with better binding properties, or that provides for immobilization of the antibody or antigen on the support without significant loss of antibody binding activity or specificity. Suitable coupling proteins include, but are not limited to, macromolecules such as serum albumins including bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH), immunoglobulin molecules, thyroglobulin, ovalbumin, and other proteins well known to those skilled in the art. Other molecules that can be used to bind antibodies the support include polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and the like. Such molecules and methods of coupling these molecules are well known to those of ordinary skill in the art and are described in, for example, U.S. Pat. No.

7,595,307, U.S. Pat. Appl. No. US 2009/0155299, the disclosures and cited references therein of which are incorporated by reference herein.

[0132] The following examples are included for purposes of illustration and are not intended to limit the scope of the invention. [00170] EXAMPLES

[00171] Synthesis of building blocks

[00172] The building blocks 3, 4, 5 and 6 can be synthesized as shown in figures 2-10 using known methods and known reagents.

Claims

1. A method for forming a beta-glucan with a beta (1 ,6) bond comprising the step of:

(a) reacting for a time sufficient to form a beta (1 ,3) linkage (i) a compound (IV)

n is 0, 1, 2, 3, 4, 5, 6, 7, or 8 and (ii) a compound (III)

(III)

where al is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;

bl is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10; wherein Kal+bl < 15;

X is a bond, a linker or a protected linker;

Y is absent, -H, a carrier or a solid support.

2. The method of claim 1 , wherein intermediate (IV) is

3. A method for forming a beta-glucan with a beta (1 ,3) bond comprising the step of:

(b) reacting for a time sufficient to form a beta (1,3) linkage (i) an intermediate (V)

m is 1, 2, 3, 4, 5, 6, 7, or 8 and (ii) a compound (III)

(III)

where al is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;

bl is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10; wherein Kal+bl < 15;

X is a bond, a linker or a protected linker;

Y is absent, -H, a carrier or a solid support.

4. The method of claim 3 wherein compound (V) is

-glucan of the formula (I):

where a is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10; bis 0, 1,2,3,4, 5, 6, 7,8,9, 10;

c is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10; wherein 2<a+b+c < 15;

d is 1, 2, 3, 4, 5; wherein each d unit (a+b+c) can be the same or different;

X is a bond or a linker;

Y is absent, -H, a carrier or a solid support. -glucan of the formula (II):

where a is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;

b is 0, 1,2,3,4, 5, 6,7, 8,9, 10;

c is 0, 1, 2, 3, 4,

5,

6, 7, 8, 9, 10; wherein 2<a+b+c < 15;

d is 1, 2, 3, 4, 5; wherein each d unit (a+b+c) can be the same or different;

X is a bond or a linker;

Y is absent, -H or a carrier,

provided that if b=0, X=a bond and Y =H, then a+b≠10 or 15 and further provided that if a=6, b=l , X=a bond and Y =H, then c≠ 2.

7. An intermediate selected from the group consisting of compound 3, 4, 13, 14, 15, 16, 26, 27, 28, 29, 30, 31, 34, 35, 36, 37, 38, 41, 42, 43, 44, and 45 as shown in Figures 2-10.