OLIGOSACCHARIDES

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 61/473,557, filed April 8, 201 1 , the entire contents of which are hereby incorporated herein by reference.

BACKGROUND

[0002] The present invention provides novel intermediates and synthetic beta-mannuronic acid oligosaccharides. The invention also provides methods for synthesizing beta-mannuronic acid oligosaccharides. The beta-mannuronic acid oligosaccharides can be conjugated to carriers and used to generate antibodies against Pseudomonas aeruginosa.

[0003] P. aeruginosa is a Gram-negative aerobic motile bacterium. It is an environmentally ubiquitous, extracellular, opportunistic pathogen that causes significant morbidity and mortality in immuno-compromised subjects. Infection is of particular significance in subjects with cystic fibrosis, burns, chronic bronchitis, bronchiectasis and cancer.

[0004] Different types of P. aeruginosa immunogens have been tried or are under development as vaccines. These include lipopolysaccharides (Hanessian et al., Nature (London) 229:209-210 (1971 ); Jones et al., Lancet ii:401 -403 (1978); Maclntyre et al., Infect. Immun. 52 (1986)), polysaccharide (Pier and Thomas, J. Infect. Dis. 148:206-213 (1983)), polysaccharide conjugate (Cryz et al., Antibiot. Chemother. 42:177-183 (1989)), outer-membrane protein (Lam et al., Infect. Immun. 42:88-98 (1983); Matthews-Greer and Gilleland, J. Infect. Dis. 155:1281 -1291

(1 987)), pilin protein (US 6,541 ,007), mucoid exopolysaccharide (Pier et al., Science 249:537-540 (1990)), flagella (Holder and Naglich, J. Trauma. 26:1 18-122 (1986); Rotering and Dorner, Antibiot. Chemother. 42:218-228 (1989)), protease (Sezen et al., Zentralbl. Bakteriol. Hyg. I. Abt. Orig.

231 :126-132 (1975)), elastase (Cryz et al., Infect. Immun. 39:1072-1079 (1983), exotoxin A (Cryz et al., Infect. Immun. 39:1072- 079 (1983); Lydick et al., J. Infect. Dis. 151 :375 (1985)), and lipoprotein I (Finke et al., Infect. Immun. 59:1251 -1254 (1991 )).

[0005] The present invention provides methods for synthesizing beta- mannuronic acid (beta-ManA) oligosaccharides, which have

immunomodulatory activity. The present invention also provides novel intermediates useful in the synthesis of beta-ManA oligosaccharides.

BRIEF SUMMARY

[0006] The present invention provides novel intermediates and synthetic beta-mannuronic acid (ManA) containing oligosaccharides. The invention also provides methods for synthesizing beta-ManA containing

oligosaccharides. In some embodiments, the beta-ManA containing oligosaccharides can be conjugated to carriers and used to generate antibodies against Pseudomonas aeruginosa.

DETAILED DESCRIPTION OF THE PRESENTLY PREFERRED EMBODIMENTS

[0007] In order to provide a clear and consistent understanding of the specification and claims, the following definitions are provided.

[0008] 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.

[0009] 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., Man), 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.

[0010] As used herein, "synthetic" refers to material which is assembled from mono- or oligosaccharide building blocks that are chemically synthesized. These building blocks are coupled together to construct the larger oligosaccharide. Synthetic material 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.

[0011] 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.

[0012] 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.

[0013] As used herein, "conjugated" refers to a chemical linkage, either covalent or non-covalent, that proximally associates an oligosaccharide with a carrier so that the oligosaccharide conjugate has increased immunogenicity relative to an unconjugated oligosaccharide.

[0014] As used herein, "conjugate" refers to an oligosaccharide chemically coupled to a carrier through a linker and/or a cross-linking agent.

[0015] 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.

[0016] 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.

[0017] As used herein, "immunoprotective composition" refers to a composition formulated to provide protective immunity in a host.

[0018] 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.

[0019] 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.

[0020] 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.

[0021] 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.

[0022] As used herein, "antigen" refers to include any substance that may be specifically bound by an antibody molecule.

[0023] As used herein, "immunogen" and "immunogenic composition" refer to an antigenic composition capable of initiating lymphocyte activation resulting in an antigen-specific immune response. [0024] 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.

[0025] Synthetic Oligosaccharides and Intermediates

[0026] The present inv ManA donor 3:

[0027] where R ¹ and R ² are each independently hydroxyl protecting groups, including for example benzyl, 4-halobenzyl, tert-butyl dimethylsilyl, triethylsilyl, triisoproylsilyl, tert-butyldiphenylmethylsilyl, methoxymethyl, and benzyloxymethyl;

[0028] R ³ is a hydroxyl protecting group different from R ¹ and R ² and includes for example, levulinoyl, acetate, monochloroacetate, 4-acetoxy- 2,2-dimethylbutanoate, propargyl, tert-butyl dimethylsilyl, triethylsilyl, triisoproylsilyl, tert-butyidiphenylmethylsilyl, methoxymethyl,

benzyloxymethyl, and tetrahydropyran; and

[0029] R ⁴ is an alkyl or aromatic group such as, for example, methyl, ethyl, and benzyl.

[0030] ManA donor 3 can be used to synthesize beta-ManA containing oligosaccharides 7, including beta-ManA oligosaccharides 5. [0031] In one embodiment, the ManA donor is of the formula 3a:

[0032] Beta-ManA containing oligosaccharides 7 contain ManA monosaccharides at least at every other site and have the formula:

HO-[Z-ManA] _m -0-R 7

[0033] where m is 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0034] Z is a monosaccharide (where each Z can be the same or different);

[0035] R is H, C-i-8 alkyl, C ₂ -8 alkenyl or is resin or is -XY;

[0036] X is a bond or a linker;

[0037] Y is H, R' or a carrier, where R' is a hydroxyl-protecting group (which can be the same or different than an R).

[0038] In oligosaccharides 7, Z can be any monosaccharide. Preferred monosaccharides are hexoses or pentoses.

[0039] In some embodiments, oligosaccharide 7 is composed of ManA subunits. Such beta-ManA oligosaccharides 5 have the formula:

where n is 2, 3, 4, 5, 6, 7, 8, 9, or 0; and R is as above.

[0040] The ManA donor can be synthesized such as exemplified below and as shown in the following scheme.

86% [0041] This ManA donor can then be used to synthesis various synthetic oligosaccharides using automated techniques such as exemplified below and as shown in the following schemes.

[0042] Scheme 1

HO-[Z-ManA] _m -0-resin + HO-ManA-[Z-ManA] _m -0- resin

[0043] A method of making an oligosaccharide, comprising reacting a compound of the formula:

[0044] where R ¹ and R ² are each independently a hydroxyl protecting group;

[0045] R ³ is a hydroxyl protecting group different from R ¹ and R ² ; and

[0046] R ⁴ is a C-i-8 alkyl or C _6- io aromatic group;

[0047] with an immobilized compound of the formula:

HO-[Z-ManA] _m -0-resin

[0048] where m is 1 , 2, 3, 4, 5, 6, 7, 8, or 9; and

[0049] each Z is independently a monosaccharide or a hydroxyl- protected monosaccharide.

[0050] Scheme 2

[0051] where n, R ¹ , R ² , R ³ , and R ⁴ are as described above.

0052] Scheme 3

n=2-10 4

[0053] In the above methods, other resins besides Merrifield can be used, including for example, SynPhase Lanterns, JandaGel, Tentagel, PEG-grafted polystyrene etc.

[0054] Procedures for coupling and decoupling the oligosaccharide to the resin are known in the art and are exemplified below for Merrifield resin. Procedures for coupling subsequent donor 3 to the forming immobilized oligosaccharide are exemplified below.

[0055] Conjugation of Synthetic Oligosaccharide

[0056] Suitable linkers for conjugation comprise at one end a grouping able to enter into a covalent bonding with a reactive functional group of the carrier, e.g. an amino, thiol, or carboxyl group, and at the other end a grouping likewise able to enter into a covalent bonding with a hydroxyl group of an oligosaccharide 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.

[0057] Linkers able to react with thiol groups on the carrier are, for example, maleimide and carboxyl groups; preferred groupings able to react with aldehyde or carboxyl groups 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.

[0058] 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.

[0059] 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).

[0060] 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.

[0061] 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.

[0062] 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).

[0063] 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. Carbohydrate 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.

[0064] CRM conjugates may be purified via size exclusion

chromatography to remove and recover any unreacted oligosaccharide. Phenol/sulfuric acid (specific for carbohydrate 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.

[0065] In another embodiment, carbohydrate antigens may be

conjugated to one or more carriers 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= 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 (Thermo) in maleimide conjugation buffer (Thermo). Conjugation may be performed at pH=7.2 to avoid hydrolysis of the maleimide group during conjugation.

[0066] The present invention further provides immunogenic and immunoprotective compositions containing a synthetic oligosaccharide 5 or ManA-containing oligosaccharide 7 and antibodies derived therefrom for diagnosing, treating, and preventing infections caused by P. aeruginosa.

[0067] The present invention contemplates the use of single- and multivalent 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 P. aeruginosa. Thus, in one embodiment, the compositions may contain a single oligosaccharide 4 or 5 or 7.

[0068] 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 oligosaccharides 4 or 5 or 7.

[0069] 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.

[0070] 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. [0071] 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 "carriers," 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.

[0072] 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; RIBI, 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- 0 (function not defined) and TLR-1 1 -(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 l: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 ISA-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 AblSCO ^® -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 (IFA); 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-(1 '2'-dipalmitoyl-n-glycero-3-hydroxyphosphoryl; SAF-1 (Syntex); AS05 (GlaxoSmith Kline, Pa,); and combinations thereof.

[0073] 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, DP 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.

[0074] In one embodiment, the present invention provides

pharmaceutically acceptable immunogenic or immunoprotective

oligosaccharide compositions and their use in methods for preventing P. aeruginosa 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 oligosaccharide 4-protein 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.

[0075] Administration of the oligosaccharide- or oligosaccharide conjugate compositions or antibodies, as described herein may be carried out by any suitable means, including by parenteral administration (e.g., intravenously, subcutaneously, intradermal^, 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. [0076] 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.

[0077] 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.

[0078] 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.

[0079] 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_.

[0080] The compositions of the present invention may be administered to any animal species at risk for developing an infection by P. aeruginosa. [0081] 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.

[0082] 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 pg to about 5,000 pg of oligosaccharide for a 70 kg patient, (e.g., 1 .0 pg, 2.0 pg, 2.5 pg, 3.0 pg, 3.5 pg, 4.0 pg, 4.5 pg, 5.0 pg, 7.5 pg, 10 pg, 12.5 pg, 15 pg, 1 7.5 pg, 20 pg, 25 pg, 30 pg, 35 pg, 40 pg, 45 pg, 50 pg, 75 pg, 100 pg, 250 pg, 500 pg, 750 pg, 1 ,000 pg, 1 ,500 pg, 2,000 pg, 2,500 pg, 3,000 pg, 3,500 pg, 4,000 pg, 4,500 pg or 5,000 pg). 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 pg to 5 pg/kg body weight.

[0083] A primary dose may optionally be followed by boosting dosages of from about .0 to about 1 ,000 μg of peptide (e.g., 1 .0 pg, 2.0 pg, 2.5 pg,

3.0 pg, 3.5 pg, 4.0 pg, 4.5 pg, 5.0 pg, 7.5 pg, 10 pg, 12.5 pg, 15 pg, 17.5 pg, 20 pg, 25 pg, 30 pg, 35 pg, 40 pg, 45 pg, 50 pg, 75 pg, 100 pg, 250 pg, 500 pg, 750 pg, 1 ,000 pg, 1 ,500 pg, 2,000 pg, 2,500 pg, 3,000 pg,

3,500 pg, 4,000 pg, 4,500 pg or 5,000 pg) 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.

[0084] 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 P. aeruginosa. 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.

[0085] Antibody Compositions

[0086] In another embodiment, the invention provides an antibody preparation against one or more oligosaccharides 4 or 5 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.

[0087] Pharmaceutical antibody compositions may be used in a method for providing passive immunity against a bacterial target species of interest, including P. aeruginosa. 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.

[0088] 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. [0089] Antibodies in diagnostic assays

[0090] 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 P. aeruginosa.

[0091] Antisera to P. aeruginosa oligosaccharide 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 (IFA). 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 oligosaccharide conjugate.

[0092] The oligosaccharides and antibodies generated therefrom can be used as diagnostic reagents for detecting P. aeruginosa 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.

[0093] 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.

[0094] Typically, a solid support is first reacted with a first binding component (e.g., an anti- P. aeruginosa antibody or P. aeruginosa oligosaccharide) 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.

[0095] The following examples are included for purposes of illustration and are not intended to limit the scope of the invention.

[0096] EXAMPLES

[0097] General experimental procedures. All chemicals were used as received unless stated otherwise. ¹ H and ¹³ C NMR spectra were recorded on a Bruker DMX-400 (low-temperature NMR experiments), Bruker AV-400

(400/100 MHz) and a Bruker DMX-600 (600/150 MHz) spectrometer.

Chemical shifts (δ) are given in ppm relative to tetramethylsilane as internal standard. Coupling constants are given in Hz. All given ¹³ C-APT spectra are proton decoupled. IR-spectra were recorded on a Shimadzu FTIR-8300. Flash chromatography was performed on Fluka silica gel 60 (0.04 - 0.063 mm). TLC-analysis was conducted on DC-alufolien (Merck, Kieselgel60, F254) with detection by UV-absorption (254 nm) where applicable and by spraying with 20% sulfuric acid in ethanol followed by charring at ~150°C or by spraying with a solution of (ΝΗ ₄ ) ₆ Μο ₇ θ ₂₄ ·Η2θ (25 g/l) and (NH ₄ ) ₄ Ce(S0 ₄ ) ₄ ^« 2H ₂ 0 (10 g/l) in 10% sulfuric acid in water followed by charring at ~150°C. TLC-MS analysis was performed on a Camag TLC-MS Interface combined with an API165 (SCIEX) mass spectrometer (eluted with tert-butylmethylether/EtOAc/MeOH, 5/4/1 , v/v/v + 0.1 % formic acid, flow rate 0.1 mL/min). The solvents used in the

automated oligosaccharide synthesis were dried on molecular sieves (4A) for 24 h. The donor was co-evaporated with toluene prior to use.

[0098] Synthesis of the donor.

[0099] Compound 1 was synthesized in eight steps starting from D- mannose according to van den Bos et al., J. Am. Chem. Soc, 128, 13066- 13067 (2006). The anomeric thio functionality was hydrolyzed using NIS/TFA to yield hemiacetal 2 in 84%. Subsequently the N- (phenyl)trifluoroacetimidoyl moiety was introduced under mild basic conditions to yield imidate donor 3 in 86%.

[00100] Methyl (4-0-levulinoyl-2,3-di-0-benzyl- α/β-D- mannopyranosyluronate) (2). A solution of compound 1 (0.74 g, 1 .28 mmol) in DCM/H20 (14.3 mL, 10/1 , v/v) was cooled to 0 °C, followed by the addition of N-iodosuccinimide (0.29 g, 1 .28 mmol) and trifluoroacetic acid (0.95 mL, 1 .28 mmol). The dark purple emulsion was stirred for 2.5 h after which time sat. aq. Na ₂ S ₂ 0 ₃ (25 mL) was added. The mixture was stirred for 30 min, diluted with EtOAc and the layers were separated. The organics were washed with sat. aq. NaHC0 ₃ (2x), dried over Na ₂ S0 and concentrated in vacuo. Purification by flash column chromatography (silica gel, 66% EtOAc in PE) gave the title compound as a yellowish oil (Yield:

0.55 g, 1 .13 mmol, 88%).TLC: R _f 0.23 (PE/EtOAc, 1/1 , v/v); IR (neat, cm ^"1 ): 696, 725, 907, 1717, 1744, 3421 ; 1 H NMR (CDCI _3l 400 MHz, HH-COSY, HSQC): 57.19-7.33 (m, 10H, CH _arom ), 5.53 (t, 1 H, J = 6.7 Hz, H-4), 5.49 (s, 1 H, H-1 ), 5.15 (bs, 1 H, 1 -OH), 4.72 (d, 1 H, J = 12.2 Hz, CHH Bn), 4.63 (d, 1 H, J = 12.2 Hz, CHHBn), 4.57 (d, 1 H, J = 12.1 Hz, CHH Bn), 4.52 (d, 1 H, J = 12.1 Hz, CHHBn), 4.45 (d, 1 H, J = 6.1 Hz, H-5), 3.93 (dd, 1 H, J = 2.8, 7.0 Hz, H-3), 3.67 (s, 1 H, H-2), 3.56 (s, 3H, CH ₃ C0 ₂ Me), 2.65 (t, 2H, J = 6.4 Hz, CH ₂ Lev), 2.48-2.54 (m, 2H, CH ₂ Lev), 2.11 (s, 3H, CH ₃ Lev); 13C- APT NMR (CDCIs, 100 MHz, HSQC): 5D206.5 (C=0 Lev), 171.4, 168.9 (C=0 C0 ₂ Me, Lev), 137.8, 137.4 (Cq), 128.0, 127.9, 127.8, 127.4, 127.3, 127.2, 127.1 (CH _arom ), 92.0 (C-1 ), 74.8 (C-2, C-3), 72.3, 71.9 (CH ₂ Bn), 70.8 (C-5), 69.1 (C-4), 52.0 (CH ₃ C0 ₂ Me), 37.3 (CH ₂ Lev), 29.4 (CH3 Lev), 27.5 (CH ₂ Lev); 13C-GATED (CDCI ₃ , 100 MHz): 692.0 (J _C i,m = 67 Hz, C-1 ); TLC-MS: m/z[M+Na+] 509.2.

[00101] Methyl (4-0-levulinoyl-2,3-di-0-benzyl-1-0-(N- [phenyl]trifluoroacetimidoyl)-a/p-D-mannopyranosyluronate) (3).

Compound 2 (1.21 g, 2.49 mmol) was dissolved in acetone/H ₂ 0 (26.2 mL, 20/1 , v/v) and the solution was cooled to 0 °C. N-

(Phenyl)trifluoroacetimidoyl chloride (0.56 mL, 3.73 mmol) and potassium carbonate (0.41 g, 2.98 mmol) were added and the resulting suspension was stirred overnight at room temperature. The mixture was diluted with EtOAc and H ₂ 0, the organic layer was collected and washed with sat. aq. NaCI (2x), dried over Na ₂ S0 ₄ and concentrated in vacuo. Purification by flash column chromatography (silica gel, 33% EtOAc in PE) yielded the title compound as a colorless oil (Yield: 1.42 g, 2.15 mmol, 86%).

Analytical data are reported for the major isomer (a). TLC: R _f 0.33

(PE/EtOAc, 2/1 , v/v); IR (neat, cm ^"1 ):694, 733, 1117, 1152, 1206, 1717, 1748; ¹ H NMR (CDCI ₃ , 400 MHz, HH-COSY, HSQC): 57.24-7.35 (m, 12H, CHarom), 7.11 (t, 1 H, J = 7.4 Hz, CH NPh), 6.78 (d, 2H, J = 7.7 Hz, CH NPh), 6.45 (bs, 1 H, H-1 ), 5.59 (t, 1 H, J = 7.4 Hz, H-4), 4.64-4.72 (m, 2H, CH ₂ Bn), 4.61 (d, 1 H, J = 12.0 Hz, CHH Bn), 4.56 (d, 1 H, J = 12.1 Hz,

CHHBn), 4.40 (d, 1 H, J= 6.9 Hz, H-5), 3.91 (dd, 1 H, J = 2.7, 7.6 Hz, H-3),

3.79 (s, 1 H, H-2), 3.66 (s, 3H, CH ₃ CO ₂ Me), 2.73 (t, 2H, J = 6.4 Hz, CH2 Lev), 2.58 (dd, 2H, J = 6.3, 11.3 Hz, CH2 Lev), 2. 7 (s, 3H, CH ₃ Lev); 13C- APT NMR (CDCI ₃ , 100 MHz, HSQC): 5D206.1 (C=0 Lev), 171.5, 167.8 (C=0 C0 ₂ Me, Lev), 143.1 (CqNPh), 142.2 (q, J = 36 Hz, C=NPh), 137.3 (Cq), 128.6, 128.3, 128.3, 128.0, 127.9, 127.8, 127.8 (CH _aro m), 124.3, 1 19.3 (CH NPh), 115.8 (q, J = 283 Hz, CF3), 94.1 (C-1 ), 74.5 (C-3), 72.9, 72.7 (C-2, C-5), 72.7 (CH ₂ Bn), 68.7 (C-4), 52.6 (CH ₃ C0 ₂ Me), 37.6 (CH ₂ Lev), 29.7 (CH ₃ Lev), 27.8 (CH ₂ Lev); 13C-GATED (CDCI ₃ , 100 MHz): 594.1 (JCI ,HI = 177 Hz, C-1 ); TLC-MS: m/z[M+Na+] 680.0.

[00102] Automated synthesis of β-ManA oligosaccharides.

[00103] Automated synthesis was performed using an oligosaccharide synthesizer provided by Ancora Pharmaceuticals (Medford MA), using a synthesizer and procedures as described in WO2010/011828.

[00104] Solutions:

[00105] Building block = compound 3 in DCM (0.068 M)

[00106] Activator = trifluoromethanesulfonic acid in DCM (0.07 M)

[00107] Deblock = hydrazine acetate in pyridine/AcOH (4/1 , v/v, 0.14 M)

[00108] Protocol A. Agitation of the resin during washing

[00109] After addition of the appropriate solvent (2-4 mL), a gas-flow is applied from the bottom of the reaction vessel (RV) for 15 s to agitate the resin suspension. Then the RV is emptied.

[00110] Protocol B. Agitation of the resin during reaction

[00111] After addition of the appropriate solvent (2-4 mL), a gas-flow is applied from the bottom of the RV for 10 s to agitate the resin suspension. Then the purging is halted and the suspension is allowed to settle for 20 s.

[00112] Protocol C. Swelling of new resin

[00113] The RV is charged with dry resin. The resin is washed with DCM (3x), alternating THF and hexane (3x), THF (1x) and DCM (3x). Every wash step involves protocol A.

[00114] Protocol D. Priming of the resin before reaction

[00115] If applicable, the chiller temperature is set to ambient. The pre- swollen resin is washed with alternating THF and hexane (3x), followed by

THF (1x) and DCM (3x). Every wash step involves protocol A. [00116] Protocol E. Coupling cycle

[00117] The resin is suspended in DCM and agitated for the time needed to prepare the addition of the building block solution. Then the RV is emptied. The building block solution (1 .5 mL) is added and the

temperature is set to -45 °C. Simultaneously, a pause of 30 min is started. When the temperature of the chiller has reached its target point, the activator solution (300 microliters) is added. Protocol B is applied during 45 min (loop = 90). Then the RV is emptied and the solution is collected in a mixture of DCM/Et3N (50/1 , v/v) and molecular sieves (3A). The resin is washed with DCM (3x) using protocol A and the solutes are similarly collected.

[00118] Protocol F. Deblock

[00119] The resin is washed with DMF (3x) using protocol A. The deblock solution (2.5 mL) is added and the resin is agitated using protocol B for 10 min (loop = 20) while the temperature is raised to +40 °C. Then the RV is emptied into the waste.

[00120] Protocol G. Priming of the resin after deblock

[00121] The temperature of the chiller is set to ambient. The resin is successively washed with DMF (3x), DCM (3x), alternating THF and hexane (6x), 0.01 M AcOH in THF (6x) and THF (3x). Every wash step involves protocol A.

[00122] Protocol H. Suspense of the resin for isolation

[00123] The resin is washed with alternating DCM and MeOH (2x), followed by a mixture of DCM/MeOH (7/1 , v/v, 2x), both employing protocol A. Then a mixture of DCM/MeOH (7/1 , v/v) is added, the resin is agitated for 15 s after which time the gas-flow was halted and the program was paused. The suspended resin is isolated and this last procedure is repeated two times.

[00124] Typical synthesis towards a tetrasaccharide.

[00125] Automated construction of the tetrasaccharide:

[00126] The RV is charged with functionalized Merrifield polystyrene (100 mg, 34 micromol) and prepared for the synthesis using protocols C and D I o- I consecutively. Then the coupling reaction is performed three times

(protocol E), followed by protocol D to prepare the resin for the deblock. The deblock reaction is performed twice (protocol F) and the resin is washed according to protocol G and D. The sequence is then repeated three times starting from the coupling reaction. After the synthesis is complete, protocol H is used to isolate the resin, which is subsequently dried in vacuo overnight.

[00127] Cleavage of the oligosaccharide products from the resin:

The dry resin (charged to a 5-mL syringe) was washed with dry DCM (4x), suspended in DCM (3 ml_) and purged with argon for 5 min. Grubbs 1 catalyst (-10 mg) was added and the resulting purple suspension was consecutively purged with argon and ethylene gas. The mixture was allowed to stand at RT overnight. Then the solution was filtered off and the remaining resin was washed with DCM (8x). The filtrates were concentrated and passed through a short column (silica gel, eluted with PE/EtOAc). After concentration in vacuo, the residue was dissolved in DCM (10 mg/ml) and treated with activated charcoal (25 mass equivalents) overnight. The suspension was filtered using a Whatman filter-containing glassfilter funnel. The resulting colorless solution was concentrated and analyzed (crude yield: -48 mg).