Prevention / treatment of Pseudomonas aeruginosa infections

Abstract

The present disclosure generally relates to methods for generating and harnessing an immune response in a subject, comprising administering to a subject compounds, compositions, antibodies, and isolated or synthetic P. aeruginosa glycans, optionally in the form of glycoconjugates, conjugated to a carrier protein. Further provided are antibodies that selectively bind to the P. aeruginosa glycans or glycoconjugates described in the present disclosure.

Description

FIELD OF THEINVENTION

[0001] This disclosure relates to the identification and synthesis of novel methylated rhamnose-containing glycans, protein-glycan conjugates, and their use as antigens and vaccines. Also disclosed are antibodies that selectively bind to the glycans and glycoconjugates. The use of such glycans, glycoconjugates, corresponding compositions, and antibodies in the treatment and prevention of Pseudomonas aeruginosa infections is discussed. Background

[0002] In recent years, major pharmaceutical companies have moved away from antibiotic research because the risks and costs associated with development are somewhat high compared to the potential benefits.[1] An alternative to new antibiotics is the development of vaccines to prevent infections caused by antibiotic-resistant bacteria.[2] Vaccines against antibiotic-resistant Pseudomonas aeruginosa have not yet reached the market, although some vaccines are in clinical trials.[3-5]

[0003] P. aeruginosa has an extensive range of glycans on its surface and, as a result, glycoconjugate vaccines are being explored [5-7], but no such vaccines are licensed for human use [5].

[0004] In addition to vaccine approaches, the therapeutic potential of antibodies against surface carbohydrate targets should also be explored. Current examples of this approach include Medimmune's targeting of Psl and Aridis' targeting of LPS O antigens [8-11]. Medimmune's bispecific antibody approach was not pursued past phase 1 trials

[12] , but the potential for DNA-encoded mAb therapy (DMab) shows promise and could alleviate the high cost of specific mAb therapeutics

[13] . Such trials suggest that antibodies against P. aeruginosa may have potential therapeutic applications. Summary of the Invention

[0005] The present disclosure generally relates to compounds and compositions and vaccines comprising novel isolated or synthetic P. aeruginosa glycan antigens, optionally conjugated to a carrier protein in the form of glycoconjugates. Antibodies that selectively bind to P. aeruginosa glycans or glycoconjugates are further provided. Also disclosed are therapeutic uses and methods, as well as methods for generating and harnessing an immune response in a subject.

[0006] In one aspect, the present invention provides a compound of formula A: α-Rha3OMe (-4α-Rha3OMe) n - Formula A 1. An antigenic compound comprising an oligosaccharide moiety of wherein n is 1 to 5, preferably 2 to 4, and two positions of each Rha3OMe sugar moiety are independently substituted with -OAc or -OH.

[0007] In one aspect, the present invention provides a compound of formula A1: α-Rha3OMe (-4α-Rha3OMe) n -X formula A1 1. An antigenic compound comprising an oligosaccharide moiety of In the formula, n is 1 to 5 (preferably 2 to 4), and X is -H or -(4α-Man3OMe). m -handle, m is 0, 1 or 2, preferably 0 or 1, and two positions of each Rha3OMe sugar moiety are independently substituted with -OAc or -OH.

[0008] In one aspect, the present invention relates to a compound in which the handle is -(CH2) z NH2 or 2-glyceraldehyde, where z is an integer selected from the group consisting of 1 to 5. In one aspect, when m is 0, the handle is -(CH2)2NH2, -(CH2)3NH2 or -(CH2)3NH2.

[0009] In one aspect, when m is 1 or 2, the handle is 2-glyceraldehyde.

[0010] In one aspect, the present invention provides a method for producing a method for treating a cancer cell comprising: α-D-Rha3OMe-4-(α-D-Rha3OMe-4)4-4-α-D-Man3OMe-2-glyceraldehyde-1d (OS2), 3-O-methyl-α-D-rhamnopyranoside-(1→4)-3-O-methyl-α-D-rhamnopyranoside-(1→4)-3-O-methyl-α-D-rhamnopyranoside-(1→4)-3-O-methyl-α-D-rhamnopyranose (pentasaccharide), 3-O-methyl-α-D-rhamnopyranoside-(1→4)-3-O-methyl-α-D-rhamnopyranoside-(1→4)-3-O-methyl-α-D-rhamnopyranose (tetrasaccharide), 3-O-methyl-α-D-rhamnopyranoside-(1→4)-3-O-methyl-α-D-rhamnopyranose (trisaccharide) [ka] The present invention provides an antigenic compound selected from the group consisting of:

[0011] In one aspect, the invention comprises a linker for attachment to a carrier protein, the linker having the formula A2: α-Rha3OMe (-4α-Rha3OMe) n -X-Linker Formula A2 An antigenic compound having the formula: In the formula, n is 1 to 5, and X is -(4α-Man3OMe). m -(handle) p -, m is 0, 1 or 2, preferably 0 or 1, p is 0 or 1, and two positions of each Rha3OMe sugar moiety are independently substituted with -OAc or -OH.

[0012] In one aspect, the present invention provides an antigenic compound comprising a conjugate of an antigenic compound conjugated to a carrier protein.

[0013] In one aspect, the conjugate has the formula: α-Rha3OMe (-4α-Rha3OMe) n -X-(linker) q -Carrier Protein Formula A3 wherein n is 1 to 5 (preferably 2 to 4) and X is -(4α-Man3OMe) m -(handle) p -, m is 0, 1 or 2, preferably 0 or 1, p is 0 or 1, q is 0 or 1, and two positions of each Rha3OMe sugar moiety are independently substituted with -OAc or -OH.

[0014] In one aspect of the invention, the carrier protein comprises CRM197, tetanus toxoid (TT), Pseudomonas aeruginosa protein, human serum albumin (HSA), bovine serum albumin (BSA), diphtheria toxin B fragment (DTFB), DTFB C8, diphtheria toxoid (DT), C fragment of TT, pertussis toxoid, cholera toxoid, E. coli LT, E. coli ST or exotoxin A from Pseudomonas aeruginosa. In one aspect, the carrier protein is CRM197, TT or Pseudomonas aeruginosa protein.

[0015] In one aspect, the invention provides a pharmaceutical composition comprising a compound or conjugate described herein and a pharma- ceutically acceptable diluent, carrier or excipient, hi one aspect, the pharmaceutical composition is a vaccine.

[0016] In one aspect, the invention provides a method of generating an immune response in a subject, the method comprising administering to the subject a compound, conjugate or pharmaceutical composition as described herein.

[0017] In one aspect, the invention provides a method of preventing a Pseudomonas aeruginosa infection in a subject, the method comprising administering to the subject a compound, conjugate or pharmaceutical composition described herein.

[0018] In one aspect the invention provides a compound, conjugate, vaccine or pharmaceutical composition for use in the prevention of Pseudomonas aeruginosa infection.

[0019] In one aspect, the invention provides an antibody or antigen-binding fragment thereof that selectively binds to a compound or conjugate described herein, P. aeruginosa LPS, and / or P. aeruginosa cells, and optionally the antibody or antigen-binding fragment thereof is a monoclonal antibody or antigen-binding fragment thereof.

[0020] In one aspect, the present invention provides an antibody or antigen-binding fragment thereof that selectively binds to isolated oxidized A band terminal epitope antigen (OS2), optionally an antibody or antigen-binding fragment thereof that is a monoclonal antibody or antigen-binding fragment thereof.

[0021] In one aspect, the invention provides an antibody or antigen-binding fragment thereof described herein, which is a chimeric or humanized antibody.

[0022] In one aspect, the invention provides an antibody, or antigen-binding fragment thereof, comprising a heavy chain variable domain comprising a variable heavy chain CDR1, a variable heavy chain CDR2, and a variable heavy chain CDR3, the variable heavy chain CDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:10, and SEQ ID NO:19; the variable heavy chain CDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:11, and SEQ ID NO:20; The present invention provides an antibody or antigen-binding fragment thereof, wherein the variable heavy chain CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:12, and SEQ ID NO:21.

[0023] In one aspect, the invention provides an antibody, or antigen-binding fragment thereof, comprising a light chain variable domain comprising a variable light chain CDR1, a variable light chain CDR2, and a variable light chain CDR3, the variable light chain CDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:15, and SEQ ID NO:24; the variable light chain CDR2 comprises an amino acid sequence selected from the group consisting of GTS and RVS; The present invention provides an antibody or antigen-binding fragment thereof, wherein the variable light chain CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:16, and SEQ ID NO:25.

[0024] In one aspect, the present invention provides an antibody or antigen-binding fragment thereof comprising a combination of a heavy chain variable domain (VH) and a light chain variable domain (VL), the combination comprising: VH comprising the amino acid sequence of SEQ ID NO: 4 and VL comprising the amino acid sequence of SEQ ID NO: 8; VH comprising the amino acid sequence of SEQ ID NO: 13 and VL comprising the amino acid sequence of SEQ ID NO: 17; and VH comprising the amino acid sequence of SEQ ID NO: 22 and VL comprising the amino acid sequence of SEQ ID NO: 26 The present invention provides an antibody or antigen-binding fragment thereof selected from the group consisting of:

[0025] In one aspect, the invention provides an antibody, or antigen-binding fragment thereof, for use in treating a Pseudomonas aeruginosa infection.

[0026] In one aspect, the invention provides an antibody, or antigen-binding fragment thereof, for use in diagnosing a Pseudomonas aeruginosa infection.

[0027] In one aspect, the invention provides a method for treating a Pseudomonas aeruginosa bacterial infection in an animal comprising administering to the animal an antibody, or antigen-binding fragment thereof, described herein.

[0028] In one aspect, the invention provides a method for the diagnosis of a Pseudomonas aeruginosa bacterial infection in an animal, comprising contacting a test sample with an antibody or antigen-binding fragment thereof described herein and detecting specific binding thereto.

[0029] In one aspect, the present invention provides a synthetic process for producing a compound of formula A1, wherein m is 0, comprising: anomerically deprotecting the 3-O-methylated rhamnopyranosides to form 3-O-methylated rhamnopyranoses; acetylating the 3-O-methylated rhamnopyranose to form an acetylated 3-O-methylated rhamnopyranoside; partially deprotecting O-4 of the acetylated 3-O-methylated rhamnopyranoside to form a deprotected acetylated 3-O-methylated rhamnopyranoside; coupling the O-4 deprotected acetylated 3-O-methylated rhamnopyranosides to form acetylated 3-O-methylated oligosaccharides; deprotecting the O-4 of the acetylated 3-O-methylated oligosaccharides to form O-4 deprotected acetylated 3-O-methylated oligosaccharides; deacetylating the partially deprotected acetylated 3-O-methylated oligosaccharide to form a compound of formula A1; The present invention provides a process including:

[0030] In one aspect, the present invention provides a synthetic process for producing a compound of formula A1, where X is a handle, comprising: glycosylation of an activated O-3 methylated rhamnopyranoside intermediate having a handle containing a protected amine at 1-O to form a protected 1-O glycoside intermediate, the activated monorhamnopyranoside intermediate containing a protecting group at the glycosylation site 4-O; removing the protecting group from 4-O to form a deprotected 1-O glycoside intermediate; coupling the deprotected 1-O glycoside intermediate to an activated O-3 methylated rhamnopyranoside intermediate, which contains a protecting group at 4-O, to form a protected methylated di-, tri-, tetra- or pentasaccharide; removing all protecting groups from the protected oligosaccharide, the oligosaccharide being preferably a trisaccharide, tetrasaccharide or pentasaccharide; The present invention provides a process including: [Brief description of the drawings]

[0031] [Figure 1] FIG. 1 shows the structures of some of the isolated compounds described herein. [Diagram 2] Figure 2 shows the H NMR spectra of band A polysaccharide obtained from LPS by alkali treatment (lower trace) and its NaIO oxidation product OS1 (upper trace). * denotes the anomeric signal of an impurity, rhamnose H-1, derived from the R-rhamnan repeating unit, Rm-3-OMe-Rha. [Diagram 3] Figure 3 shows the overlap of the gCOSY spectrum (70% gray), TOCSY spectrum (50% gray), and ROESY spectrum (black) of A-PS (anomeric region) isolated from P. aeruginosa PAO1 wzy mutant by mild acid hydrolysis of LPS. [Figure 4] Figure 4 shows the positive ion mode ESI-MS spectrum of OS1. The two peaks labeled m / z 1117.8 and 1122.6 correspond to the ammonium and sodium adducts of Rha5Man1 tetritol-1d1Me6. [Diagram 5] 5A and 5B show 1H NMR spectra of L-monosaccharides (FIG. 5A) and D-monosaccharides (FIG. 5B) recorded by CD3OD. [Figure 6] FIG. 6 shows the GC-MS traces of the produced OS1 octyl glycoside and standard substances. [Figure 7] FIG. 7 shows the structure of the P. aeruginosa A-band 3-O-methyl-D-rhamnose pentasaccharide chip bound to 3-O-methyl-D-mannose and glyceraldehyde prior to conjugation to CRIM. [Figure 8] Figures 8A, 8B, 8C, and 8D show MALDI-MS analyses of BSA (Figure 8A), BSA-3-O-methyl rhamnan conjugate (Figure 8B), CRM (Figure 8C), and CRM-3-O-methyl rhamnan conjugate (Figure 8D). [Figure 9]9A and 9B show ELISA titrations of IgM (left) and IgG (right) antibodies of D56 serum from mice (M1-M6) immunized with CRM-3-O-methyl rhamnan conjugate against BSA-3-O-methyl rhamnan conjugate (upper curve) and PAO1 wtLPS (lower curve) (FIG. 9A), or ELISA titrations of D72 serum from rabbits (R1-R2) immunized with CRM-3-O-methyl rhamnan conjugate against PAO1 wtLPS (left) and BSA-3-O-methyl rhamnan conjugate (right) (FIG. 9B). [Figure 10] FIG. 10 shows ELISA analysis of binding of 1B1, 3B8, and 3C4 mAb-containing hybridoma supernatants (used undiluted) to purified LPS antigen from P. aeruginosa strains PAO1wt, PAO1(wzy::Gm)(Δpa5457), and PAO1(wzy::Gm)(Δpa5458). [Figure 11] Figures 11A, 11B, and 11C show ELISA analysis of binding of purified monoclonal antibodies (mAbs) of 1B1 (Figure 11A), 3B8 (Figure 11B), and 3C4 (Figure 11C) (all at 100 μg / ml) to killed whole cells of P. aeruginosa strains NRCC#6678, 6668-70, 6954-60 and negative control strains M. catarrhalis 6541 and N. meningitidis 6263 (full strain details see Table 1). [Figure 12] Figure 12 shows ELISA analysis of binding of purified mAbs 3C4 (panel a), 3B8 (panel b), and 1B1 (panel c) (all at 100 μg ml-1) to killed whole cells of clinical isolates of P. aeruginosa strains NRCC#6678 and 6944-53 (full strain details see Table 1). [Figure 13] 13A and 13B show opsonophagocytosis assay titration curves of mAb 1B1 (solid line), mAb 3B8 (dashed line), and mAb 3C4 (dotted line) against P. aeruginosa strain PAO1 BAA-47 (FIG. 13A) and P. aeruginosa strain 537 (FIG. 13B). [Figure 14]FIG. 14A shows a competitive ELISA between mAb 1B1 and mAb 3C4 against purified PAO1 LPS. mAb 1B1 was titrated on an ELISA plate coated with PAO1 LPS at the dilutions shown on the x-axis. The plate was washed and mAb 3C4 was added at a fixed concentration (62.5 μg / ml). A secondary mAb specific for mAb 3C4 was added and absorbance was measured after adding a colorimetric reagent (line with square markers). The absorbance obtained when 3C4 was added to the plate without competition from 1B1 is shown by the line with triangular markers. FIG. 14B shows a competitive ELISA between mAb 3B8 and mAb 3C4 against purified PAO1 LPS. mAb 3B8 was titrated on an ELISA plate coated with PAO1 LPS at the concentrations shown on the x-axis. The plate was washed and mAb 3C4 was added at a fixed concentration (62.5 μg / ml). A secondary mAb specific for mAb 3C4 was added and absorbance was measured after addition of a colorimetric reagent (line with square markers). The absorbance obtained when 3C4 was added to the plate without competition from 3B8 is shown by the line with triangle markers. [Figure 15]15A, 15B, and 15C show inhibition ELISAs of mAb 1B1 (FIG. 15A), mAb 3C4 (FIG. 15B), and mAb 3B8 (FIG. 15C) against P. aeruginosa PAO1 BAA-47 LPS using LPS (left hand panels; square marker-P. aeruginosa PAO1 BAA-47, inverted triangle marker-P. aeruginosa (wzy::Gm)(Δpa5458), diamond marker-N. meningitidis galE / lpt3, and circle marker-PBS control) or synthetic oligosaccharides representing terminally methylated rhamnan (right hand panels; filled circle marker-pentasaccharide, open square marker-tetrasaccharide, open circle marker-trisaccharide, inverted triangle marker-disaccharide, diamond marker-disaccharide with linker, triangle marker-L monomer, filled square marker-D monomer). Equal volumes of serial dilutions of either LPS or synthetic oligosaccharides, as indicated on the x-axis, were mixed with 10 μg / ml mAb. After 1 h of incubation, 100 μl of this inhibition mixture was added to PAO1 BAA-47 LPS-coated plates. A secondary mAb specific for the primary mAb was added and absorbance was measured after addition of a color reagent. The absorbance obtained when the mAb was no longer inhibited by additional LPS or synthetic oligosaccharides was equivalent to inhibition by PBS. Figures 15D, 15E, and 15F show inhibition ELISAs of mAb 1B1 (Figure 15D), mAb 3C4 (Figure 15E), and mAb 3B8 (Figure 15F) against P. aeruginosa PAO1 BAA-47 whole cells using LPS (left hand panels; square marker-P. aeruginosa PAO1 BAA-47, inverted triangle marker-P. aeruginosa (wzy::Gm)(Δpa5458), diamond marker-N. meningitidis galE / lpt3, and circle marker-PBS control) or synthetic oligosaccharides representing terminally methylated rhamnan (right hand panels; filled circle marker-pentasaccharide, open square marker-tetrasaccharide, open circle marker-trisaccharide, inverted triangle marker-disaccharide, diamond marker-disaccharide with linker, triangle marker-L monomer, blue-D monomer). Equal volumes of serial dilutions of either LPS or synthetic oligosaccharides, as indicated on the x-axis, were mixed with 10 μg / ml mAb.After 1 hour of incubation, 100 μl of this inhibition mixture was added to the PAO1 BAA-47 whole cell coated plate. Secondary mAbs specific for the primary mAb were added and absorbance was measured after addition of color reagent. The absorbance obtained when the mAb was no longer inhibited by additional LPS or synthetic oligosaccharides was equivalent to inhibition by PBS. [Figure 16] In Figure 16 we present SPR sensorgrams showing the binding of synthetic oligosaccharides to a high density 1B1 IgM surface. Different concentration ranges of synthetic oligosaccharides (see Experimental) were run over IgM 1B1 and an irrelevant IgM, Fn 4F1. Kinetics and affinities are reported in Table 5. Mcat lgt2 / 4: control oligosaccharide. [Figure 17] FIG. 17 shows the 1H NMR spectrum of the disaccharide compound (600 MHz, CD3OD). [Figure 18] FIG. 18 shows the 1H NMR spectrum of the trisaccharide compound (500 MHz, CD3OD). [Figure 19] FIG. 19 shows the 1H NMR spectrum of the tetrasaccharide compound (500 MHz, CD3OD). [Figure 20] FIG. 20 shows the 1H NMR spectrum of the pentasaccharide compound (600 MHz, CD3OD). [Figure 21] FIG. 21 shows the 1H NMR spectrum of the disaccharide compound (600 MHz, DO). [Figure 22] FIG. 22 shows a reaction scheme for the synthesis of D-monosaccharides (Scheme 1). [Figure 23] FIG. 23 shows a reaction scheme for the synthesis of L-monosaccharides (Scheme 2). [Figure 24] FIG. 24 shows a reaction scheme for the synthesis of 3-O-methyl D-rhamnose oligosaccharides (Scheme 3). [Diagram 25] FIG. 25 shows the reaction scheme for the addition of an aminoethyl handle to a disaccharide (Scheme 4). [Figure 26]Figure 26A shows SDS-PAGE (left) of HSA (lane 2) and HSA-3-O-methyl rhamnan pentasaccharide conjugate (two concentrations, lanes 3 and 4), and Figure 26B shows Western blot (using mAb 1Bl; right) analysis of HSA (lane 2) and HSA-3-O-methyl rhamnan pentasaccharide conjugate (two concentrations, lanes 3 and 4). Molecular weight markers are shown in lane 1. [Figure 27] FIG. 27 shows MALDI-MS analysis of a) HSA, b) HSA-3-O-methyl rhamnan pentasaccharide conjugate. [Figure 28] Figures 28A, 28B, 28C, 28D, 28E, 28F, 28G, and 28H show ELISA for the ability to recognize P. aeruginosa LPS using mouse sera from synthetic conjugate immunization. Titration of IgM and IgG antibodies at various time points is shown. [Figure 29] FIG. 29 shows ELISA of antisera from synthetic oligosaccharide conjugates against killed whole cells. [Diagram 30] FIG. 30 shows reaction schemes for the synthesis of tri-, tetra-, and pentasaccharides containing a handle and optionally a linker. [Figure 31A] FIG. 31A shows the NMR spectrum of 3-O-methyl-α-D-rhamnopyranoside-(1→4)-3-O-methyl-α-D-rhamnopyranoside-(1→4)-2-(2,2-dimethoxybutylcarbonyl)amino]ethyl 3-O-methyl-α-D-rhamnopyranoside (S22), 1H NMR, 600 MHz, CD3OD. [Figure 31B] FIG. 31B shows the NMR spectrum of 3-O-methyl-α-D-rhamnopyranoside-(1→4)-3-O-methyl-α-D-rhamnopyranoside-(1→4)-3-O-methyl-α-D-rhamnopyranoside-(1→4)-2-(2,2-dimethoxybutylcarbonyl)amino]ethyl 3-O-methyl-α-D-rhamnopyranoside (S23), 1H NMR, 600 MHz, CD3OD. [Diagram 32]FIG. 32 shows the NMR spectrum of 3-O-methyl-α-D-rhamnopyranoside-(1→4)-3-O-methyl-α-D-rhamnopyranoside-(1→4)-3-O-methyl-α-D-rhamnopyranoside-(1→4)-3-O-methyl-α-D-rhamnopyranoside-(1→4)-2-(2,2-dimethoxybutylcarbonyl)amino]ethyl 3-O-methyl-α-D-rhamnopyranoside (S24), 1H NMR, 600 MHz, CD3OD. [Diagram 33] Figure 33 shows inhibition ELISA of mAb 1B1 against P. aeruginosa PAO1 BAA-47(wt) LPS using LPS (left hand graph; P. aeruginosa PAO1 BAA-47(wt); N. meningitidis galE / lpt3; PBS control) or synthetic oligosaccharides with linkers representing terminally methylated rhamnans (right hand graph; pentasaccharide; tetrasaccharide; trisaccharide). Serial dilutions are shown on the x-axis. [Figure 34A] Figure 34A shows an ELISA determining recognition of BSA-oligosaccharide conjugates by pre-immune mouse serum IgM (1:40 dilution) prior to CRM-oligosaccharide conjugate immunization. MRha3V 1-5 mice were administered the trisaccharide CRM conjugate, MRha4V 6-10 mice were administered the tetrasaccharide CRM conjugate, and MRha5V 11-15 mice were administered the pentasaccharide CRM conjugate. [Figure 34B] Figure 34B shows an ELISA to determine recognition of Pa wtLPS by pre-immune mouse serum IgM titers prior to CRM-oligosaccharide conjugate immunization. MRha3V 1-5 mice were administered the trisaccharide CRM conjugate, MRha4V 6-10 mice were administered the tetrasaccharide CRM conjugate, and MRha5V 11-15 mice were administered the pentasaccharide CRM conjugate. [Figure 35A] Figure 35A shows an ELISA to determine recognition of BSA-trisaccharide conjugates by terminal bleed mouse serum IgM titers after CRM-oligosaccharide conjugate immunization. MRha3V 1-5 mice were administered the trisaccharide CRM conjugate, MRha4V 6-10 mice were administered the tetrasaccharide CRM conjugate, and MRha5V 11-15 mice were administered the pentasaccharide CRM conjugate. [Figure 35B] Figure 35B shows an ELISA to determine recognition of the BSA-tetrasaccharide conjugate by terminal bleed mouse serum IgM titers after CRM-oligosaccharide conjugate immunization. MRha3V 1-5 mice were administered the trisaccharide CRM conjugate, MRha4V 6-10 mice were administered the tetrasaccharide CRM conjugate, and MRha5V 11-15 mice were administered the pentasaccharide CRM conjugate. [Figure 35C] Figure 35C shows an ELISA to determine recognition of the BSA-pentasaccharide conjugate by terminal bleed mouse serum IgM titers after CRM-oligosaccharide conjugate immunization. MRha3V 1-5 mice were administered the trisaccharide CRM conjugate, MRha4V 6-10 mice were administered the tetrasaccharide CRM conjugate, and MRha5V 11-15 mice were administered the pentasaccharide CRM conjugate. [Figure 35D] Figure 35D shows an ELISA to determine recognition of LPS by terminal bleed mouse serum IgM titers after CRM-oligosaccharide conjugate immunization. MRha3V 1-5 mice received the trisaccharide CRM conjugate, MRha4V 6-10 mice received the tetrasaccharide CRM conjugate, and MRha5V 11-15 mice received the pentasaccharide CRM conjugate. [Figure 36A] Figure 36A shows an ELISA determining recognition of BSA-oligosaccharide conjugates by pre-immune mouse serum IgG (1:40 dilution) prior to CRM-oligosaccharide conjugate immunization. MRha3V 1-5 mice were administered the trisaccharide CRM conjugate, MRha4V 6-10 mice were administered the tetrasaccharide CRM conjugate, and MRha5V 11-15 mice were administered the pentasaccharide CRM conjugate. [Figure 36B] Figure 36B shows an ELISA to determine recognition of Pa wtLPS by pre-immune mouse serum IgG titers prior to CRM-oligosaccharide conjugate immunization. MRha3V 1-5 mice were administered the trisaccharide CRM conjugate, MRha4V 6-10 mice were administered the tetrasaccharide CRM conjugate, and MRha5V 11-15 mice were administered the pentasaccharide CRM conjugate. [Figure 37A]Figure 37A shows an ELISA determining recognition by final bleed mouse serum IgG (1:20 dilution) after CRM-oligosaccharide conjugate immunization to BSA-oligosaccharides and Pa wtLPS as indicated. MRha3V 1-5 mice were administered the trisaccharide CRM conjugate, MRha4V 6-10 mice were administered the tetrasaccharide CRM conjugate, and MRha5V 11-15 mice were administered the pentasaccharide CRM conjugate. Pre-immune serum (Pre) was used as a negative control and mAb 3C4 (isotype IgG2b) was used as a positive control for Pa wtLPS. [Figure 37B] Figure 37B shows ELISAs determining recognition of pooled pre-immune mouse serum IgG and terminal bleed mouse serum IgG (1:40 dilution) after CRM-oligosaccharide conjugate immunization against Pa wtLPS, Pa wzy5457LPS, Pa wzy5458LPS, and Nm wtLPS (negative control) as indicated. MRha3V 1-5 mice were administered trisaccharide CRM conjugates, MRha4V 6-10 mice were administered tetrasaccharide CRM conjugates, and MRha5V 11-15 mice were administered pentasaccharide CRM conjugates. Pre-immune serum (Pre) was used as a negative control. [Figure 38A] Figure 38A shows an ELISA determining recognition of BSA-oligosaccharide conjugates by pre- and post-immunization (D70) rabbit sera (1:500 and 1:1500 dilutions) before and after CRM-oligosaccharide conjugate immunization. RRha3V 1-2 rabbits were administered the trisaccharide CRM conjugate, RRha4V 3-4 rabbits were administered the tetrasaccharide CRM conjugate, and RRha5V 5-6 rabbits were administered the pentasaccharide CRM conjugate. [Figure 38B] Figure 38B shows ELISA to determine recognition of Pa wtLPS, Pa wzy5457LPS, and Pa wzy5458LPS by pre- and post-immunization (D70) rabbit serum titrations before and after CRM-oligosaccharide conjugate immunization. RRha3V 1-2 rabbits were administered the trisaccharide CRM conjugate, RRha4V 3-4 rabbits were administered the tetrasaccharide CRM conjugate, and RRha5V 5-6 rabbits were administered the pentasaccharide CRM conjugate. [Figure 39A] FIG. 39A shows ELISA analysis of binding of pooled pre- and post-immune mouse sera (IgM and IgG all diluted 1:40) to killed whole cells of P. aeruginosa strains NRCC#6678, 6667-70, 6954-60, and the negative control strain M. catarrhalis 6541 (full strain details see Table 7). [Figure 39B] FIG. 39B shows ELISA analysis of binding of individual pre- and post-immune rabbit sera (all diluted 1:1500) to killed whole cells of P. aeruginosa strains NRCC#6678, 6667-70, 6954-60, and the negative control strain M. catarrhalis 6541 (see Table 7 for full strain details). [Figure 39C] FIG. 39C shows ELISA analysis of binding of individual pre- and post-immune rabbit sera (all diluted 1:1500) to killed whole cells of P. aeruginosa clinical strains NRCC#6678, 6944-46, 6948-53 and negative control strains Neisseria meningitidis 6263 and M. catarrhalis 6541 (see Table 7 for full strain details). Detailed Description

[0032] The present invention is based in part on the identification of a novel P. aeruginosa polysaccharide structure by NMR and chemical analysis. The structure provided herein is believed to be the first to identify, i.e., the first to precisely identify, the P. aeruginosa A-band terminal epitope antigen (A-PS).

[0033] Pseudomonas aeruginosa (also called P. aeruginosa) is an opportunistic bacterial pathogen and the causative agent of several potentially life-threatening infections, including healthcare-associated and ventilator-associated pneumonia, chronic lung infections in cystic fibrosis (CF) patients, and burn and soft tissue infections. An increasing prevalence of drug-resistant P. aeruginosa infections has been observed. Thus, in some instances, the term "P. aeruginosa" refers to pathogenic strains of P. aeruginosa, including but not limited to antibiotic-resistant strains, such as P. aeruginosa strains resistant to beta-lactam antibiotics (e.g., penicillin), piperacillin, imipenem, tobramycin, or ciprofloxacin. In some embodiments, the term "P. aeruginosa" refers to pathogenic strains that infect cystic fibrosis patients.

[0034] The term "infection" refers to any microbial infection of a subject's body. An infection involves the invasion of a patient's body by a microorganism and the subsequent replication of the microorganism in the subject's body. In a particular example, the microorganism is Pseudomonas aeruginosa.

[0035] The term "subject," as used herein, refers to an animal and may include, for example, domestic animals, such as cats, dogs, etc., livestock (e.g., cows, horses, pigs, sheep, goats, etc.), laboratory animals (e.g., mice, rabbits, rats, guinea pigs, etc.), mammals, non-human mammals, primates, non-human primates, rodents, birds, reptiles, amphibians, fish, and any other animals. In a particular example, the subject is a human.

[0036] Glycan As used herein, the terms "carbohydrate," "glycan," "sugar," "oligosaccharide," and "polysaccharide" are used interchangeably and refer to an oligomer or polymer composed of sugar monomers, typically linked by glycosidic bonds, also referred to herein as bonds. In a glycan, the monosaccharide monomers may all be the same or may be different. Common monomers include, but are not limited to, triose, tetraose, pentose, glucose, fructose, galactose, rhamnose and 3-O-methyl rhamnose, xylose, arabinose, lyxose, allose, altrose, mannose and 3-O-methyl mannose, gulose, iodose, ribose, mannoheptulose, sedoheptulose, and talose. In a glycan, amino sugars may also be monomers. Glycans containing such sugars are referred to herein as aminoglycans. An amino sugar, as used herein, is a sugar molecule that contains an amine group instead of a hydroxy group, or in some embodiments, a sugar derived from such a sugar. Examples of amino sugars include, but are not limited to, glucosamine, galactosamine, N-acetylglucosamine, N-acetylgalactosamine, sialic acid (including, but not limited to, N-acetylneuraminic acid and N-glycolylneuraminic acid), and L-daunosamine.

[0037] In some cases, the glycan may be modified with one or more non-glycan components, including, but not limited to, labels, handles, linkers, spacers, carriers, etc. In some embodiments, the glycan may comprise a glycoconjugate. A glycoconjugate may include, but is not limited to, a glycoprotein, a glycolipid, or a proteoglycan. A glycoprotein includes any protein (glycan) that contains covalently attached oligosaccharide chains. Unless otherwise specified, polysaccharide nomenclature used herein follows the IUB-IUPAC Joint Commission on Biochemical Nomenclature (JCBM) Recommendations 1980. See JCBN, 1982, J. Biol. Chem. 257:3352-3354.

[0038] The label "D-monosaccharide" as used herein, e.g. in the Figures, specifically refers to 3-O-methyl-D-rhamnose, the label "L-monosaccharide" as used herein, e.g. in the Figures, specifically refers to 3-O-methyl-L-rhamnopyranose and the label "disaccharide" as used herein, e.g. in the Figures, specifically refers to 3-O-methyl-α-D-rhamnopyranosido-(1→4)-3-O-methyl-D-rhamnopyranose, as will be clear from the context.

[0039] The term "antigen" as used herein refers to a substance capable of initiating and mediating an immune response. The immune response stimulated by an antigen may be either humoral or cellular, or both, and is generally specific for the antigen. An antigen that stimulates or enhances an immune response is said to be immunogenic and may be called an immunogen. A composition that contains an antigen may be called an "antigenic composition" or an "immunogenic composition."

[0040] Thus, an antigen is a substance to which an antibody molecule or a T cell receptor can bind. Many types of biomolecules and other molecules can act as antigens. For example, antigens can be derived from molecules including, but not limited to, proteins, peptides, carbohydrates, polysaccharides, oligosaccharides, sugars, lipids, phospholipids, glycophospholipids, and other molecules, as well as fragments and / or combinations thereof.

[0041] Antigens may be derived from innate sources or from sources exogenous to a particular mammal or other animals (e.g., infectious agents). Antigens may have multiple antigenic determinants such that exposure of a mammal to the antigen may result in multiple corresponding antibody or cellular immune responses with different specificities.

[0042] As noted above, in particular examples, isolated or chemically synthesized glycan antigens derived from P. aeruginosa are described.

[0043] Antigens may act to sensitize the host by presenting antigens in association with MHC molecules on the cell surface. In addition, antigen-specific T cells or antibodies may be produced to allow for subsequent protection of the immunized host. Thus, the immunogenic composition may protect the host from bacterial infection and reduce the severity, i.e., death, from bacterial infection. Antigens may also be used to generate polyclonal or monoclonal antibodies, which may be used to confer passive immunity to subjects. Antigens may also be used to generate functional antibodies, as measured by bacterial killing, either by animal efficacy models or opsonophagocytic injury assays.

[0044] As used herein, the term "isolated" in relation to polysaccharides refers to the isolation of the A-band terminal epitope antigen (A-PS) from purified polysaccharides using purification techniques known in the art, including the use of centrifugation, depth filtration, sedimentation, ultrafiltration, treatment with activated charcoal, diafiltration, and / or column chromatography. In general, isolation of polysaccharides refers to the partial removal of proteins, nucleic acids, and non-specific endogenous polysaccharides. Isolated polysaccharides contain less than 10%, 8%, 6%, 4%, or 2% protein impurities and / or nucleic acids.

[0045] As used herein, the term "purification" in relation to bacterial polysaccharides refers to the purification of the polysaccharides from cell lysates by means such as centrifugation, sedimentation, and ultrafiltration. In general, purification of polysaccharides refers to the removal of cell debris and DNA.

[0046] Bacterial glycans can be derived from naturally occurring bacteria, genetically modified bacteria, or synthetically produced. Polysaccharides are typically subjected to one or more processing steps prior to use, such as purification, functionalization, depolymerization using mildly acidic or oxidative conditions, deacetylation, etc. Post-processing steps can also be used as needed. Any method known in the art suitable for synthesis, preparation, and / or purification, suitable polysaccharides, and oligosaccharides can be utilized.

[0047] P. aeruginosa produces a variety of cell surface glycans. Previous studies have identified a consensus polysaccharide (PS) antigen, often referred to as A-band PS, which is composed of a repeating neutral D-rhamnan trisaccharide unit as a relatively conserved cell surface carbohydrate. One study showed no immunogenic effect of the neutral D-rhamnan trisaccharide

[25] . As mentioned above, the inventors have identified a novel P. aeruginosa polysaccharide structure and shown that the carbohydrate antigen consists of an immunogenic methylated rhamnan oligosaccharide at the non-reducing end of A-band PS. α-D-Rha3OMe-4-(α-D-Rha3OMe-4-)4-

[0048] In particular, the inventors isolated and characterized A-PS1 and A-PS2 (see FIG. 1). It is believed that the structures provided herein are the first to identify, and thus the first to accurately identify, the P. aeruginosa A band terminal epitope antigen. This A-PS chip (see A-PS1 and A-PS2 in FIG. 1) was further separated into antigenic components to produce OS1 and OS2. It was determined that the methylated rhamnose moiety confers antigenicity. Di-, tri-, tetra-, and penta-3OMe rhamnose oligosaccharides were synthesized with and without a handle at the reducing end.

[0049] Thus, in one aspect, the present invention provides a compound of formula A: α-Rha3OMe (-4α-Rha3OMe) n - Formula A wherein n is 1-5, preferably 2-4, and two positions of each Rha3OMe sugar moiety are independently substituted with -OAc or -OH.

[0050] In one aspect, the present invention provides a compound of formula A1: α-Rha3OMe (-4α-Rha3OMe) n -X formula A1 wherein n is 1 to 5 (preferably 2 to 4), and X is -H or -(4α-Man3OMe). m -handle, m is 0, 1 or 2, preferably 0 or 1, and two positions of each Rha3OMe sugar moiety are independently substituted with -OAc or -OH.

[0051] The sugar monomer moieties can independently be in the D or L configuration.

[0052] In some cases, the 2-O of the sugar moiety may be independently acetylated. In other cases, an alternating pattern of acetylated and non-acetylated 3-O-methyl rhamnose may be used. In other cases, the 2-O may be replaced with groups other than acetate, such as glycolyl and lactyl, to improve immunogenicity.

[0053] Specific examples of methylated rhamnose oligosaccharides include: α-D-Rha3OMe-4-(α-D-Rha3OMe-4)4-4-α-D-Man3OMe-2-glyceraldehyde-1d (OS2), 3-O-methyl-α-D-rhamnopyranoside-(1→4)-3-O-methyl-α-D-rhamnopyranoside-(1→4)-3-O-methyl-α-D-rhamnopyranoside-(1→4)-3-O-methyl-α-D-rhamnopyranose (pentasaccharide), 3-O-methyl-α-D-rhamnopyranoside-(1→4)-3-O-methyl-α-D-rhamnopyranoside-(1→4)-3-O-methyl-α-D-rhamnopyranose (tetrasaccharide) (III), 3-O-methyl-α-D-rhamnopyranoside-(1→4)-3-O-methyl-α-D-rhamnopyranose (trisaccharide) 3-O-methyl-α-D-rhamnopyranoside-(1→4)-3-O-methyl-α-D-rhamnopyranoside-(1→4)-3-O-methyl-α-D-rhamnopyranoside-(1→4)-3-O-methyl-α-D-rhamnopyranoside-(1→4)-3-O-methyl-α-D-rhamnopyranoside-(1→4)-4-3-O-methyl-α-D-mannopyranoside Examples include: [ka]

[0054] The methylated rhamnose oligosaccharides of the present invention can be isolated or chemically synthesized, or the isolated oligosaccharides can be further chemically modified.

[0055] Isolation of A-band terminal epitope antigen: The inventors have developed a method for isolating A-band terminal epitope antigen (A-PS). In one aspect, a method is provided for producing A-band terminal epitope antigen (OS1) comprising the step of subjecting isolated LPS (lipopolysaccharide) from P. aeruginosa to acid or alkaline hydrolysis to produce a high molecular weight fraction. In one example, the high molecular weight fraction is subjected to acid hydrolysis, for example, with acetic acid. In one example, the high molecular weight fraction is subjected to alkaline hydrolysis, for example, by reaction with KOH, followed by treatment with HCl.

[0056] The macromolecular fraction is then subjected to an oxidation step followed by acid hydrolysis to produce OS1. In one example, this first oxidation step includes reacting the macromolecular fraction with NaIO4, reacting with ethylene glycol and NaBD4, reacting with AcOH, and desalting to produce a product, which is subjected to acid hydrolysis to produce an isolated A-band terminal epitope antigen (OS1). In a particular example, the product has the formula: α-D-Rha3OMe-4-(α-D-Rha3OMe-4)4-4-α-D-Man3OMe-2-tetritol-1d(OS1) This invention describes an isolated A-band terminal epitope antigen (OS1) from Pseudomonas aeruginosa, having the following structure:

[0057] When OS1 is subjected to a further oxidation step, a further isolated A-band terminal epitope is obtained, identified herein as OS2. In one example, this second oxidation step includes reacting OS1 with NaIO4, reacting with ethylene glycol and NaBD4, reacting with AcOH, and desalting to produce a product, which is subjected to acid hydrolysis to produce an isolated A-band terminal epitope antigen (OS2). In a particular example, the formula: α-D-Rha3OMe-4-(α-D-Rha3OMe-4)4-4-α-D-Man3OMe-2-glyceraldehyde-1d(OS2) We describe an isolated oxidized A-band terminal epitope antigen (OS2) from Pseudomonas aeruginosa, which is a glycan of the compound.

[0058] In some instances, the compound is an antigen.

[0059] Chemical Synthesis: The chemical synthesis of the compounds of the invention is described in detail in the Examples and illustrated in FIGS.

[0060] In one aspect, there is provided a synthetic process for producing a compound of the invention, comprising: anomerically deprotecting the 3-O-methylated rhamnopyranosides to form 3-O-methylated rhamnopyranoses; acetylating the 3-O-methylated rhamnopyranose to form an acetylated 3-O-methylated rhamnopyranoside; partially deprotecting O-4 of the acetylated 3-O-methylated rhamnopyranoside to form a deprotected acetylated 3-O-methylated rhamnopyranoside; coupling the O-4 deprotected acetylated 3-O-methylated rhamnopyranosides to form acetylated 3-O-methylated oligosaccharides; deprotecting the O-4 of the acetylated 3-O-methylated oligosaccharides to form O-4 deprotected acetylated 3-O-methylated oligosaccharides; deacetylating the partially deprotected acetylated 3-O-methylated oligosaccharide to form a compound; A process is provided that includes:

[0061] In one aspect, there is provided a synthetic process for producing a compound of the invention, comprising: [ka] A process is provided that includes:

[0062] Handle: The compounds of the present invention also include compounds that include a "handle". A "handle", in the context of the present invention, is a chemical modification at a site distal to the terminal α-Rha3OMe repeat sequence to form a reactive group. Examples include 2-glyceraldehyde, -CH2-NH2, -(CH2)2NH2, -(CH2)3NH2, -(CH2)4NH2, (CH2)5NH2, -C(O)OH, -C(O)H, -C(O)NH2, and -CH2N3. When the compound includes -4α-Man3OMe (i.e., m is 1 or 2), the handle is preferably 2-glyceraldehyde. When the compound does not contain 4α-Man3OMe (i.e., m is 0), the handle is preferably -CH2-NH2, -(CH2)2NH2, -(CH2)3NH2, -(CH2)4NH2, (CH2)5NH2, -C(O)OH, -C(O)H, -C(O)NH2, and -CH2N3, more preferably -CH2-NH2, -(CH2)2NH2, -(CH2)3NH2, -(CH2)4NH2 or -(CH2)5NH2.

[0063] In one example, the synthesis process includes the following: glycosylation of an activated O-3 methylated rhamnopyranoside intermediate having a handle containing a protected amine at 1-O to form a protected 1-O glycoside intermediate, the activated monorhamnopyranoside intermediate containing a protecting group at the glycosylation site 4-O; removing the protecting group from 4-O to form a deprotected 1-O glycoside intermediate; coupling the deprotected 1-O glycoside intermediate to an activated O-3 methylated rhamnopyranoside intermediate, which contains a protecting group at 4-O, to form a protected methylated di-, tri-, tetra- or pentasaccharide; removing all protecting groups from the protected disaccharide, trisaccharide, tetrasaccharide, or pentasaccharide; The method further includes the step of adding the handle by performing:

[0064] In one example, the synthesis process includes the following: [ka] The method further comprises the step of attaching a handle to a compound of the invention by performing

[0065] Linkers: Purified or synthetic oligosaccharides may optionally contain a linker that can be conjugated to the oligosaccharides of the invention directly or via a handle on the oligosaccharide. As will be appreciated by those skilled in the art, the linker may be any suitable linker for the desired purpose, for example, for conjugating 4α-linked glycans to a carrier protein. Suitable linkers containing functional groups at both ends, such as acids or NHS esters or PFP esters, are known to those skilled in the art and include, but are not limited to, polyethylene glycol (PEG), linear polyamidoamines (PAA), poly(2-oxazolines) (POx), poly(glycerol adipate) (PGA), polyhydroxyalkanoic acid (PHA), and other linkers suitable for the preparation of glycoconjugates, such as those described in Munneke et al.

[42] . Examples of suitable linkers are available, for example, as BCN PEG and BCN reagents from BroadPharm®.

[0066] In certain embodiments, an oligosaccharide can be coupled to a linker to form a polysaccharide linker in which the free terminus of the linker is an ester group. Thus, the linker is one in which at least one terminus is an ester group. The other terminus is selected so that it can react with an oligosaccharide to form an oligosaccharide linker intermediate.

[0067] In certain embodiments, the linker is a bifunctional linker that generates a first ester group for reacting with a primary amine group on the oligosaccharide handle and a second ester group for reacting with a primary amine group in the carrier molecule. An exemplary linker is adipic acid N-hydroxysuccinimide diester (SIDEA). In one aspect, the linker may be a bifunctional linker that includes an activated N-hydroxysuccinimide and a hemiacetal protected aldehyde, one end of which is reacted with a primary amine on the oligosaccharide handle.

[0068] Other suitable techniques use carbodiimides, hydrazides, active esters, norbornane, p-nitrobenzoic acid, N-hydroxysuccinimide, S-NHS, EDC, TSTU. Many are described in WO 98 / 42721. Conjugation may involve a carbonyl linker that may be formed by reacting the free hydroxy group of the sugar with CDI (see Bethell et al.,

[43] ) followed by reaction with the protein to form a carbamate bond. This conjugation may involve reducing the anomeric terminus to a primary hydroxy group, optionally protecting / deprotecting the primary hydroxy group, reacting the primary hydroxy group with CDI to form a CDI carbamate intermediate, and coupling the CDI carbamate intermediate to an amino group on the protein.

[0069] In some cases, the linker may be used to attach at the anomeric position. In some cases, the linker may be placed early in the synthesis process. It may be placed on a disaccharide, trisaccharide, tetrasaccharide, or pentasaccharide, or in addition to the acetylated version and non-acetylated / acetylated pattern of such sugars. In other cases, it may be possible to attach the oligosaccharide to any binding functionality for potential conjugation to proteins.

[0070] In one aspect, the linker can be, for example, amino-peg, and the functional groups can be, for example, azide and aldehyde protecting groups.

[0071] In one example, the linker may be added according to the following process. [ka]

[0072] Conjugates: Conjugation to carrier proteins can improve immunogenicity. Suitable classes of proteins include pilins, outer membrane proteins, and excretory toxins of pathogenic bacteria, non-toxic or "toxoid" forms of such toxins, non-toxic proteins that are antigenically similar to bacterial toxins (i.e., cross-reactants or CRMs), and other proteins. In one embodiment, a CRM, such as CRM197, can be used as a carrier protein. CRM197 is a non-toxic variant of diphtheria toxin (DT). Other suitable carrier proteins include further inactivated bacterial toxins, such as DT, diphtheria toxoid B fragment (DTFB), DTB C8, TT (tetanus toxoid) or C fragment of TT, pertussis toxoid, cholera toxoid, E. coli LT (heat-labile enterotoxin), E. coli ST (heat-stable enterotoxin), and Pseudomonas proteins, such as exotoxin from P. aeruginosa. Also included are human serum albumin (HSA) and bovine serum albumin (BSA).Other DT variants, such as CRM176, CRM228, CRM45; CRM9, CRM45, CRM102, CRM103, CRM3201, and CRM107, can also be used as carrier proteins.Also included are Clostridium perfringens exotoxins / toxoids.Other suitable bacterial proteins include, but are not limited to, pneumococcal surface protein A (PspA), pneumococcal adhesin protein (PsaA), and pneumococcal surface proteins BVH-3 and BVH-11. The use of immunogenic carrier proteins from non-mammalian sources, including keyhole limpet hemocyanin (KLH), horseshoe crab hemocyanin, and plant edestin, is also contemplated, as is the use of viral proteins such as hepatitis B surface / core antigens, rotavirus VP7 protein, and respiratory syncytial virus F and G proteins. Other carrier proteins will be known to those of skill in the art.

[0073] Glycan conjugates can be prepared by known coupling techniques. The oligosaccharides of the invention can be conjugated to carrier proteins to form glycoconjugates, either directly or via handles and / or linkers, using the chemical techniques discussed above with respect to linkers.

[0074] Conjugation between the glycan and the carrier can be achieved using a variety of reagents. Conjugation can be performed directly between the glycan and the carrier protein, such as direct covalent binding by reductive amination. Alternatively, conjugation can be performed using a cross-linking agent.

[0075] In one example, a protein can be conjugated to a glycan-handle-linker compound as follows: [ka]

[0076] Further examples of protein and linker conjugations are provided below.

[0077] In some examples, the antigen is a natural sugar (OS2) extracted and modified from Pseudomonas aeruginosa, which can be conjugated to a carrier protein, e.g., CRM, HSA, BSA, etc. In this case, glyceraldehyde acts as a handle and the protein is attached via a reductive amination reaction. [ka]

[0078] In some examples, the antigen comprises or consists of: [ka]

[0079] In some cases, direct reductive amination of the hemiacetal with a protein may be used as an example. In some cases, this is accomplished via the reducing end of a synthetic antigen. In some cases, the linker may first be added to the sugar via a handle and conjugated to the attached linker via known coupling techniques.

[0080] In one aspect, the carrier protein or linker may be conjugated by direct reductive amination with an amine from the carrier protein or linker, respectively (R is the protein or linker). The following are examples: [ka]

[0081] In some instances, the 2-O of the oligosaccharide may be acetylated. [ka]

[0082] In other instances, a combination of non-acetylated and acetylated monosaccharides may be used, while in other instances, an alternating pattern of acetylated and non-acetylated 3-O-methyl rhamnose may be used. [ka]

[0083] Although specific compounds are exemplified above, the chemical techniques also apply to other oligosaccharides of the invention, including the corresponding pentasaccharides, tetrasaccharides, trisaccharides, and disaccharides.

[0084] Pharmaceutical / vaccine compositions The present invention further provides compositions, including pharmaceutical compositions, immunogenic compositions, and vaccine compositions, that comprise, consist essentially of, or consist of any of the glycans described herein, including both glycans conjugated to a carrier protein and unconjugated glycans, together with a pharma- ceutically acceptable carrier, excipient, and / or adjuvant.

[0085] Formulation can be achieved using methods recognized in the art. For example, the glycan can be formulated with a physiologically acceptable vehicle to prepare the composition. Examples of such vehicles include, but are not limited to, water, buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol), and dextrose solutions.

[0086] In some aspects, compositions are described that include a conjugate and a pharma- ceutically acceptable excipient. In some aspects, compositions are described that include a conjugate, a pharma- ceutically acceptable excipient, and an adjuvant.

[0087] In some examples, vaccines are described in which a glycan is an antigenic component of the vaccine.

[0088] The term "vaccine" as used herein refers to a substance used to stimulate the production of antibodies and / or provide immunity against one or more diseases, prepared from a disease-causing agent, its product or a synthetic substitute, and processed to act as an antigen without inducing disease.

[0089] A vaccine typically includes an antigen that resembles the disease-causing agent or is generated from an attenuated or killed form of the disease-causing agent. For example, a vaccine may have one or more antigens from a bacterium, one or more of its surface proteins, or one or more of its membrane components. A vaccine may be prophylactic (to reduce the risk of developing or reverse the effects of future infections with natural or "wild" pathogens) or therapeutic (e.g., a vaccine against a disease or disorder under investigation). In some embodiments, the vaccines described herein are prophylactic vaccines. In some embodiments, the vaccines described herein are therapeutic vaccines. In some embodiments, a vaccine may be both a prophylactic and a therapeutic vaccine.

[0090] Initial tests carried out with isolated antigens allowed the generation of conjugates used to immunize mice and rabbits to generate monoclonal and polyclonal antibodies. The polyclonal antibodies were able to recognize the majority of P. aeruginosa strains in our collection, and one of the three monoclonal antibodies generated was able to recognize the majority of P. aeruginosa strains and facilitate their opsonophagocytic killing. This monoclonal antibody was able to recognize most of the P. aeruginosa strains in our collection, including clinical and serotype strains.

[0091] Adjuvants can be used to induce an enhanced immune response in a subject. Thus, adjuvants for use with the present invention can be selected based on their ability to affect antibody titers.

[0092] The term "adjuvant" as used herein refers to any substance that acts to promote, prolong or enhance an antigen-specific immune response when used in combination with a particular vaccine antigen. An adjuvant can be a naturally occurring component contained in an attenuated or killed immunogen. For example, an adjuvant can be a whole cell, a protein or protein fragment, or a component of the lipid membrane of a bacterial cell. An adjuvant can also be a synthetic compound. For example, an adjuvant can be an aluminum salt, a phospholipid, or a derivative thereof.

[0093] In general, adjuvanted vaccines can help induce stronger local as well as systemic immune responses compared to non-adjuvanted vaccines.

[0094] In some examples, the method of generating an immune response further comprises administering an adjuvant.

[0095] In some cases, water-in-oil emulsions may be useful as adjuvants. Water-in-oil emulsions may act by forming mobile antigen depots, facilitating sustained release of antigens, and enhancing antigen presentation to immune components. Freund's adjuvants may be used as complete Freund's adjuvant (CFA), which contains dried and inactivated mycobacterial particles, or as incomplete Freund's adjuvant (IFA), which does not contain such particles. Other water-in-oil based adjuvants may include EMULSIGEN®, which contains micron-sized oil droplets that are free of animal-based components, and may be used alone or in combination with other adjuvants, including but not limited to aluminum hydroxide and CARBIGEN®.

[0096] In another example, immunostimulatory oligonucleotides may also be used as adjuvants. Such adjuvants may include CpG oligodeoxynucleotides (ODNs). CpG ODNs are recognized by Toll-like receptor 9 (TLR9) and cause strong immunostimulatory effects. C-type CpG ODNs induce strong IFN-α production and B cell stimulation of plasmacytoid dendritic cells (pDCs), as well as IFN-γ production of helper T (Tx) cells. CpG ODN adjuvants have been shown to significantly enhance pneumococcal polysaccharide (types 19F and 6B)-specific IgG2a and IgG3 in mice. CpG ODNs also enhance antibody responses to protein carrier CRM197, particularly CRM197-specific IgG2a and IgG3. In addition, immunization of old mice with pneumococcal capsular polysaccharide serotype 14 (PPS14) mixed with CpG-ODN has been shown to restore IgG anti-PPS14 responses to young adult levels. The CpG ODN used in accordance with the present invention may include class A, B, or C ODN. In some embodiments, the ODN may include any of the commercially available ones, such as ODN-1585, ODN-1668, ODN-1826, ODN-2006, ODN-2007, ODN-2216, ODN-2336, ODN-2395, and / or ODN-M362. In some cases, ODN-2395 may be used. ODN-2395 is a class C CpG ODN that significantly stimulates human TLR9 as well as mouse TLR9. Such ODNs include a phosphorothioate backbone and a CpG palindrome motif.

[0097] In some examples, in preparing a vaccine according to the present disclosure, the glycan is covalently attached or otherwise conjugated to an immunogenic carrier molecule. Typically, the immunogenic carrier molecule is a protein or polypeptide.

[0098] The term "excipient" or "pharmaceutically acceptable excipient" as used herein refers to any substance that is mixed with the compounds and / or compositions of the present invention prior to use. In some embodiments, the excipient is inert and is used primarily as a carrier, diluent or vehicle for the compounds and / or compositions of the present invention. An excipient is pharmaceutically acceptable if it is physiologically compatible, i.e., does not produce harmful or untoward reactions when administered to animals, including humans or non-human animals, as appropriate.

[0099] Formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such methods of preparation include bringing into association the active ingredient with an excipient and / or one or more other accessory ingredients.

[0100] Pharmaceutical compositions according to the disclosure may be prepared, packaged, and / or sold in bulk, as a single unit dose, and / or as a plurality of single unit doses.

[0101] Pharmaceutically acceptable excipients used in the manufacture of pharmaceutical compositions include, but are not limited to, inert diluents, dispersing and / or granulating agents, surface-active and / or emulsifying agents, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and / or oils. Such excipients may optionally be included in the pharmaceutical composition.

[0102] Examples of diluents include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate, lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, corn starch, powdered sugar, and the like, and / or combinations thereof.

[0103] The vaccines described herein are preferably capable of preventing, ameliorating, and / or treating an infection caused by P. aeruginosa in a subject.

[0104] As used herein, the terms "prevent" and "preventing" include prevention of recurrence, spread, or onset of a P. aeruginosa infection. It is not intended to limit the disclosure as being completely preventative. In some embodiments, prevention includes delaying the onset or reducing the severity of an infection.

[0105] The terms "treatment", "treat" or "treating" as used herein refer to obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results may include, but are not limited to, alleviation or amelioration of one or more symptoms or symptoms, reduction in the extent of disease, stabilization of disease state (i.e., not worsening), prevention of disease spread, delay or slowing of disease progression, amelioration or alleviation of disease state, reduction of disease recurrence, and remission (whether partial or total), detectable or not. "Treating" and "treatment" may also mean prolonging survival compared to expected survival in the absence of treatment. Thus, the terms "treatment" and "treating" are not limited to cases where a subject (e.g., patient) is cured and the disease is eradicated. Rather, examples of the present disclosure also contemplate treatment that reduces symptoms and / or delays disease progression.

[0106] The term "symptom" of a disease or disorder (eg, infection with Pseudomonas aeruginosa) is any pathological phenomenon or deviation from normal structure, function, or sensation experienced by a subject that is indicative of disease.

[0107] The term "amelioration" or "ameliorates," as used herein, refers to the reduction, decrease or elimination of a condition, disease, disorder or phenotype, including an abnormality or symptom.

[0108] P. aeruginosa is a significant opportunistic pathogen that causes a variety of life-threatening infections in immunosuppressed or immunocompromised patients. Individuals at risk for developing P. aeruginosa infections include cystic fibrosis patients, burn patients, severely neutropenic patients (e.g., cancer patients undergoing chemotherapy), and intensive care unit patients receiving respiratory support.

[0109] The vaccines described herein may be administered simultaneously with other existing vaccines.

[0110] The vaccine of the present invention may be administered to a subject by any route, including intramuscular, subcutaneous, intradermal, oral, inhalation, intranasal, rectal, and intravenous. Oral administration may suitably be administered by tablet, capsule, or suspension or emulsion. Alternatively, the vaccine may be administered via Dischaler® or Turbohaler® in the form of fine powder or aerosol. Intranasal administration may suitably be administered in the form of fine powder or aerosol nasal spray or modified Dischaler® or Turbohaler®. Rectal administration may suitably be administered by suppository.

[0111] An immunoprotective amount of the vaccine may be administered in a single dose or in a series of doses. If more than one dose is administered, the doses may be administered several days, weeks or months apart. In some cases, the vaccine may be administered as a single dose or in a series of doses, including one or more boosters.

[0112] The dosage and administration schedule of the vaccine to be administered to a subject may be determined according to standard techniques well known to those skilled in the pharmaceutical and veterinary arts, taking into account factors such as the intended use, the particular antigen, adjuvant (if any), age, sex, weight, species, general condition, medical and / or treatment history, and route of administration.

[0113] In some instances, a therapeutically effective amount of the vaccine or immunogenic composition is used.

[0114] A therapeutically effective amount refers to an amount of a particular substance sufficient to achieve a desired effect in a subject treated with that substance. For example, this may be the amount of a vaccine useful for inducing an immune response and / or preventing an infectious disease in a subject. The effective amount of a vaccine (or immunogenic composition) useful for resisting, preventing, ameliorating, and / or treating an infectious disease in a subject depends, for example, on the subject being treated, the method of administration of the therapeutic composition, and other factors.

[0115] antibody In some examples, methods are provided for generating antibodies specific to the glycan antigens described herein.

[0116] In some cases, antibodies can be generated by immunizing a host with a particular antigen. As discussed above, such an immune response typically causes an organism to produce one or more antibodies against a foreign substance, such as an antigen or a portion of an antigen. In some cases, the method of immunization can be modified based on the desired immunization result or results. As used herein, the term "immunization result" refers to the desired effect or effects of immunization. Examples include high antibody titers and / or improved antibody specificity against the target of interest. Methods for harvesting antibodies are known in the art.

[0117] As used herein, the term "antibody" is used broadly to specifically encompass a variety of embodiments including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies formed from at least two intact antibodies, for example), and antibody fragments, e.g., diabodies, so long as they exhibit the desired biological activity. Antibodies are primarily amino acid-based molecules, but may also include one or more modifications, e.g., with sugar moieties, linkers, detectable labels, and the like.

[0118] The term "monoclonal antibody," as used herein, refers to an antibody obtained from a population of substantially homogenous cells (or clones), i.e., except for possible variants that may arise during the generation of the monoclonal antibody and which are generally present in minor amounts, the individual antibodies comprising the population are identical and / or bind to the same epitope. In contrast to polyclonal antibody preparations, which typically include different antibodies specific for different determinants (epitopes), each monoclonal antibody is specific for a single determinant on the antigen.

[0119] In some examples, the antibodies described herein may be humanized antibodies.

[0120] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. Mostly, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient antibody are replaced by residues from a hypervariable region of an antibody of a non-human species (donor antibody) such as mouse, rat, rabbit or non-human primate having the desired specificity, affinity, and capacity.

[0121] The methods of the invention are conveniently practiced by providing the compounds and / or compositions used in such methods in the form of a kit. Such kits preferably include the compositions described herein. Such kits preferably include instructions for their use.

[0122] In order to obtain a better understanding of the invention described herein, the following examples are set forth. It should be understood that such examples are for illustrative purposes only, and therefore should not be construed as limiting the scope of the invention in any way.

[0123] Methods and Uses In general, the present specification describes a method for generating an immune response in a subject, the method comprising administering to the subject an isolated or synthetic glycan, preferably an isolated or synthetic glycan antigen derived from Pseudomonas aeruginosa, more preferably an isolated or synthetic glycan antigen derived from Pseudomonas aeruginosa bound to a carrier protein in the form of a glycoconjugate.

[0124] In some aspects, a method is provided for treating a subject having, suspected of having, or at risk of developing a P. aeruginosa infection, comprising administering to the subject an isolated or synthetic glycan, preferably an isolated or synthetic glycan antigen derived from P. aeruginosa, more preferably an isolated or synthetic glycan antigen derived from P. aeruginosa bound to a carrier protein in the form of a glycoconjugate.

[0125] In some aspects, a method is provided for treating a subject having, suspected of having, or at risk of developing a P. aeruginosa infection, comprising administering an antibody or derivative thereof specific for a glycan antigen from P. aeruginosa identified herein.

[0126] Generally, methods are described herein using an antibody, such as a monoclonal antibody described herein, to diagnose or treat a P. aeruginosa infection. In one aspect, the antibody is the 1B1 MAb described herein.

[0127] In one aspect, the antibodies or antigen-binding fragments thereof may be used for the treatment of a Pseudomonas aeruginosa infection.

[0128] In one aspect, the antibodies or antigen-binding fragments thereof may be used in the diagnosis of P. aeruginosa infection.

[0129] In one aspect, a method is provided for treating a P. aeruginosa infection, the method comprising administering an antibody or antigen-binding fragment to a subject.

[0130] In one aspect, a method is provided for the diagnosis of a Pseudomonas aeruginosa bacterial infection in an animal, the method comprising contacting a test sample with an antibody or antigen-binding fragment thereof and detecting specific binding thereto. EXAMPLES

[0131] P. aeruginosa produces a variety of cell surface glycans. Previous studies identified a common polysaccharide (PS) antigen, often referred to as A-band PS, which is composed of a neutral D-rhamnan trisaccharide repeat unit as a relatively conserved cell surface carbohydrate. However, the inventors have discovered novel epitopes not previously identified. Nuclear magnetic resonance (NMR) spectroscopy and chemical analysis of A-PS preparations have shown the presence of several additional components. In fact, the carbohydrate antigen consists of immunogenic methylated rhamnan oligosaccharides at the non-reducing end of A-band PS. Initial studies performed with the isolated antigens allowed the generation of conjugates used to immunize mice and rabbits to generate monoclonal and polyclonal antibodies. The polyclonal antibodies were able to recognize the majority of P. aeruginosa strains tested, and one of the three monoclonal antibodies generated was able to recognize the majority of P. aeruginosa strains and facilitate this opsonophagocytic killing. This monoclonal antibody was able to recognize all P. aeruginosa strains in our collection, including clinical and serovar strains. Synthetic oligosaccharides (mono- to pentasaccharides) representative of the terminal 3-O-methyl-D-rhamnan were prepared and a trisaccharide was identified as the minimal epitope required for efficient mimicking of the native antigen recognized by the broadly cross-reactive monoclonal antibody. Conjugation of the synthetic pentasaccharide with a carrier protein generated polyclonal antibodies in mice. Such antibodies were also able to recognize whole cells of the P. aeruginosa strains tested, further highlighting the ability of the synthetic antigen to efficiently mimic the native antigen. These experiments demonstrate the utility of such novel antigens and corresponding antibodies as vaccines, therapeutic targets and in methods of treating Pa infections.

[0132] [Table 1]

[0133] Example 1A: Isolation and oxidation of A-PS The following method was used to isolate the A-band terminal epitope: in addition to isolation from LPS, it was isolated directly from cells. The A-PS yield by EDTA extraction in this case was higher compared to isolation from LPS, but purification was more difficult using EDTA extraction.

[0134] A-band PS was isolated from PAO1 Wzy::Gm (band B OPS-free mutant) LPS by acetic acid hydrolysis followed by size-exclusion chromatography. The PS fraction was then purified by anion exchange or reverse-phase HPLC. Yields were typically low, about 5 mg A-PS from 100 mg LPS, and scale-up was not proportional to increasing amounts of starting LPS.

[0135] Alternatively, A-PS was extracted from the same mutant cells by EDTA. This method has been reported previously for the extraction of LPS, but this method did not extract significant amounts of LPS. Three main components were found in the preparation: A-PS, psl-polysaccharide with the structure described previously

[15] , and cyclic phosphorylated glucan

[16] . The signal of the cyclic glucan in the spectrum from the total mixture was not well visualized, but after anion exchange separation of the fraction eluted at high salt concentration, the A band and psl-polysaccharide were obtained in pure form. The NMR spectrum of the isolated compound showed no signals that could be attributed to lipids or other components of LPS. Thus, at least a portion of the band A rhamnan did not bind to LPS, as previously suggested

[17] . The A-PS yield by EDTA extraction in this case was much higher compared to isolation from LPS, but purification was more difficult.

[0136] A-PS was purified on a reversed-phase HPLC column or a Sep-Pak C18 cartridge, which is completely retained in water and can be eluted with 30% ethanol. All components of A-PS, including those that give minor anomeric and methyl signals in the NMR spectrum, were eluted together. In C18 column chromatography, A-PS was eluted with a wide methanol gradient starting from about 20% MeOH using a water-methanol gradient, and no peaks were visible.

[0137] A-PS was partially retained on the anion-exchange column eluted with a NaCl gradient in some fractions because it contains acidic components, a behavior that may indicate binding of A-PS to the phosphorylated, acidic LPS core.

[0138] Growth of P. aeruginosa strains: P. aeruginosa strain PAO1(wzy::Gm) (NRCC6667), known to be free of B-band LPS, was grown to isolate A-band polysaccharide (PS). 100 μg mL -1 One Difco Brain Heart Infusion (BHI) agar plate streaked with 100 mg L of gentamicin sulfate was incubated in a Forma incubator at 37°C. After 7 hours of incubation, all plate growth material was suspended in 1.5 mL of BHI broth and vortexed thoroughly to suspend the material. -1 The suspension was equally inoculated onto ten BHI agar plates supplemented with gentamicin sulfate at 100 mg L−1, spread using the hockey stick method, and incubated at 37°C. -1 Two 1L / 4L baffled flasks with BHI broth supplemented with 100 ml of gentamicin sulfate were inoculated with the products from 10 plates after 16 hours of incubation. The flasks were incubated for 2 hours at 37°C and 200 RPM in a Forma shaking incubator. When the A600nm reached an optical density of 2, these flask cultures were cultured at 100 mg L -122 L of BHI broth supplemented with 100 ml of gentamicin sulfate was inoculated in a 30 L fresh MBR (MultipleBioreactors) fermenter. Dissolved oxygen was controlled at 15% saturation by variable airflow, agitation speed, and oxygen mixing as required. After 8 3 / 4 hours of growth, the temperature was reduced to 10°C until harvest. After 22 hours, A600nm reached 13.4 and pH was 8.35. The culture was concentrated to 4 L using a Millipore tangential flow Pellicon harvester (3 x 0.22 μ membranes). The 4 L of concentrated cells were injured by the addition of 90 ml of phenol solution 95% (w / v) and placed in a Forma shaking incubator at 10°C with shaking at 175 RPM for 4 hours. A viability check of the material was performed after 3 washes with PBS. The 4 L concentrate was centrifuged in a Sorval RC6+ centrifuge at 11K RPM for 30 minutes to pellet the cells. A gelatinous, watery pellet with a wet weight of 2767 g was obtained and distributed into three 1 L containers. One container was centrifuged for an additional 30 minutes at 11K RPM to give a final wet weight of 1110 g. The cells were lyophilized and stored for later use.

[0139] Several other strains were also grown and used in this study, detailed in Table 1. After incubations were set up and grown as described above, total biomass was harvested from the 24 L fermenter.

[0140] Isolation of A-PS by EDTA extraction of whole cells: 20 g of dried cells of P. aeruginosa strain PAO1(wzy::Gm) (NRCC6667) were stirred in 10% (w / v) disodium EDTA for 1 h at 25°C. The solution was dialyzed and acetic acid was added to a final concentration of 10% to precipitate nucleic acids. The precipitate was removed by centrifugation and the solution was dialyzed and lyophilized. The material was dissolved in water and ultracentrifuged and the resulting clear solution was separated on a Biogel P6 column. The high molecular weight fraction was collected and separated by anion exchange chromatography.

[0141] Isolation of A-PS from LPS: LPS was isolated from P. aeruginosa cells from strain PAO1(wzy::Gm) (NRCC6667) as previously described

[14] . LPS (100 mg) was hydrolyzed with 2% acetic acid (100 °C, 2 h). The resulting solution was centrifuged to remove the precipitate and separated on a Biogel P6 column. The high molecular weight fraction was collected and separated by anion exchange chromatography.

[0142] Periodate oxidation: High molecular weight fractions from both above isolates (10 mg each time) were dissolved in water (2 mL) and NaIO4 (20 mg) was added. After the solution was kept in the dark at RT for 24 h, ethylene glycol (0.2 mL) and excess NaBD4 were added, the solution was kept for another 1 h, neutralized with 0.2 mL AcOH and desalted on a Sephadex G-15 column. The product was hydrolyzed with 2% AcOH for 2 h at 100 °C and separated on a Sephadex G-15 column to give OS1. For the generation of OS2 from OS1, a similar method was followed, whereby OS1 was oxidized with periodate, the reaction was quenched with ethylene glycol without borohydride reduction and purified on a Sephadex G-15 column. The structures of OS1 and OS2 are shown in Figure 1.

[0143] Gel chromatography: Gel chromatography was performed on a Sephadex G-15 column (1.5 x 60 cm) or a Biogel P6 column (2.5 x 60 cm) in 1% acetic acid and monitored by a refractive index detector (Gilson).

[0144] Anion exchange chromatography: Up to 50 mg of sample was poured into a HiTrapQ column (Amersham, two columns of 5 mL each connected together) in water for 3 mL min. -1The sample was injected at 1000 rpm and washed with water for 5 min, then eluted with a linear gradient from water to 1 M NaCl over 1 h with UV detection at 220 nm and spot checking on a silica TLC plate, developed by immersion in 5% H2SO4 in ethanol and heating with a heat gun until a brown spot was visible. Carbohydrate-positive samples were pooled and desalted with 1% AcOH on a Sephadex G-15 column and monitored by a refractive index detector.

[0145] Example 1B: Structural characterization of immunodominant epitopes of the Pseudomonas aeruginosa (Pa) A-band rhamnan chip Determination of neutral and amino sugars as alditol acetates: Samples (0.2–1 mg) were hydrolyzed with 3 M TFA (120 °C, 3 h), dried, reduced with NaBD4, and the excess of reagent was discarded with 0.5 mL of AcOH. The solution was dried under air flow and twice after addition of MeOH (1 mL) and acetylated with 0.2 mL of Ac2O with 0.2 mL of pyridine at 100 °C for 30 min. The reaction mixture was dried and analyzed on a ThermoTrace1310 instrument using an ITQ1100 ion trap detector, capillary column HP-5, 160–260 °C for 4 min. -1 GC-MS analysis was performed.

[0146] Absolute configuration: Samples and prepared standards (approximately 0.5 mg) were added with (R)- or (RS)-2-octanol (0.2 mL) and acetyl chloride (20 μL) at RT and heated (100° C., 2 h) in a sealed vial. The reaction mixture was dried by airflow, acetylated (0.2 mL Ac2O with 0.2 mL pyridine, 100° C., 30 min), dried, and then analyzed by GC-MS as described above.

[0147] NMR spectroscopy: NMR experiments were performed using a Bruker AVANCE III 600 MHz ( 1 H) A 5 mm Z-gradient probe was used on the spectrometer to measure the acetone internal standard ( 1 2.225 ppm for H and 13C 31.45 ppm) using the standard pulse sequences cosygpprqf (gCOSY), mlevphpr (TOCSY, mixing time 120 ms), roesyphpr (ROESY, mixing time 500 ms), hsqcedetgp (HSQC), hsqcetgpml (HSQC-TOCSY, 80 ms TOCSY delay), and hmbcgplpndqf (HMBC, 100 ms long-range transfer delay). Resolution was kept below 3 Hz / pt for F2 in proton-proton correlations and below 5 Hz / pt for F2 in H-C correlations. Spectra were processed and analyzed using the Topspin2.1 program from Bruker.

[0148] Monosaccharides were identified by cross peak patterns from COSY, TOCSY, and NOESY, as well as 13C NMR chemical shifts. The positions of amino groups were concluded from the upfield positions of the aminated carbon signals (CH is 45-60 ppm). The bonds between monosaccharides were determined from NOE correlations and HMBC correlations by glycosidic exchange.

[0149] Mass spectrometry: ESI MS was obtained using a Waters SQ Detector2 instrument. Samples were injected in 50% MeCN with 0.1% TFA.

[0150] Structure: P. aeruginosa A-PS consists of a D-rhamnose trisaccharide repeating unit: -2-α-Rha-3-α-Rha-3-α-Rha-A-PS repeat unit A.B.C. Contains the main component consisting of.

[0151] The NMR spectrum of the A-PS preparation contained the signals of the repeating units as well as additional signals (Figure 2). Some of the minor anomeric signals have previously been identified as belonging to 3-O-methyl-rhamnose

[18] , with a signal of the O-methyl group at approximately 3.3 ppm, but the complete structure has not been determined.

[0152] Polysaccharides obtained by EDTA extraction from LPS had similar NMR spectra. Minor differences existed, but the complex nature of the spectra did not allow identification of such minor components and discrimination of impurities from the actual components of A-PS. Because A-PS is presumed to bind to the LPS core, it was predicted that A-PS obtained from LPS should contain core components, but these could not be identified with certainty.

[0153] Monosaccharide analysis of A-PS (GC-MS with alditol acetate) showed the presence of Rha as the major component, along with small amounts of Man, Glc, 2-O-methyl-Rha, 3-O-methyl-Rha (approximately 5-8 times higher than the other minor components), 2-O-methyl-Man, and 3-O-methyl-Man. No heptose derivatives were detected.

[0154] Periodate oxidation of A-PS followed by NaBD4 reduction, mild hydrolysis, and separation of the products on a Sephadex G-15 column allowed the isolation of the expected d-rhamnan oxidation product α-d-Rha-3-α-d-Rha-2-Gro1d and the oligosaccharide OS1 (Fig. 1). OS1 was characterized by NMR and ESI-MS. Interpretation of the 2D NMR spectrum of OS1 (Table 2, Fig. 3) allowed the identification of several overlapping spin systems of a homo-oligomer of terminal 3-O-methyl-α-Rha residues (E) and 4-substituted 3-O-methyl-α-Rha residues (F). The number of residues in this oligomer was not clear from the NMR data, but MS established the presence of five 3-O-methyl-Rha residues (Fig. 4). The next residue after the 3-OMe rhamnan oligosaccharide in the glycan of OS1 was represented by 3-O-methyl-α-Man (L), which was then bound to O-2 of 1-deuterated erythritol X. 3-O-Methyl-Man was identified by GC-MS (alditol acetate) using K. pneumoniae O5OPS as a standard source

[19] . Erythritol could only be generated by oxidation-reduction of 4-substituted hexopyranose. The position of the methyl group was estimated from NOE and HMBC data and a large downfield shift of the 13C signal at the methylation position.

[0155] [Table 2]

[0156] To allow an unambiguous determination of the absolute configuration of 3-OMe-rhamnose, as both isomers of rhamnose have been identified in P. aeruginosa LPS

[20] , synthetic standards of D- and L-rhamnose monosaccharides were prepared. NMR analysis confirmed the authenticity of such standards (Fig. 5A and 5B). The absolute D-configuration of 3-O-methyl-Rha was determined by GC-MS of the acetylated 2-octanol derivative of OS1 in comparison with the identical derivative obtained from the synthetic standard (Fig. 6).

[0157] Positive mode ESI MS of the OS1 compound showed two peaks at m / z 1117.8 and 1122.6 in a 1:1 ratio, corresponding to the ammonium and sodium adducts of Rha5Man1tetritol-1d1Me6, with a calculated accurate mass of 1099.5 (Figure 4). Thus, OS1 contains five residues of 3-O-methyl-d-Rha linked by 1-4 bonds.

[0158] Similar oligosaccharides have been suggested previously

[18] , but in that study xylose was detected instead of 3-O-methyl-α-Man, and the linkage site was not identified.

[0159] Interpretation of the NMR spectra of intact A-PS confirmed the signals of the rhamnan trisaccharide repeat -ABC- (data not shown) and also indicated the presence of less intense spin systems corresponding to intact OS1, with two residues G and H identified as 2-O-methyl-Man (G) and 2-O-Me-Rha (H) (Table 3). The spectra always contained two weak spin systems of terminal α-Glc(N,N'), assumed to be Glc from the P. aeruginosa LPS core, but such residues did not generate interpretable NOEs.

[0160] [Table 3]

[0161] The 3-O-methyl-ManL of A-PS was linked to O-4 of α-ManM, and thus the erythritol X of OS1 was derived from oxidized ManM. Several spin systems of α-Man were present, which formed the trisaccharide-4-α-Man-4-α-Man-4-α-Man-(MKD) and were linked to either 2-O-methyl-RhaH or 2-O-methyl-ManG. Further tracing of the chain was not possible, as no other components could be identified. The linkage between the D-rhamnan trisaccharide repeat unit and the methylated oligosaccharides was not identified.

[0162] Since all signals of OS1 maintained the same position in A-PS and OS1, it can be concluded that oligomers of 3-O-methyl-Rha occupy the non-reducing ends of A-PS chains and, due to this terminal position, may be immunogenic.

[0163] As described herein, the detailed structure of the non-reducing end of A-band PS has been determined and it has been found that rather than simply containing a neutral rhamnan trisaccharide repeat unit, the polymer is capped with a pentasaccharide of 3-O-methyl rhamnose residues linked to the neutral mannose trisaccharide via a methylmannose molecule. Although the point of attachment to the neutral rhamnan has not been established, the fact that identical signals for 3-O-methyl Rha residues maintain identical positions in A-PS and OS1 strongly suggests that oligomers of 3-O-methyl-Rha occupy the non-reducing end of the A-PS chain. Others have observed signals consistent with 3-O-methyl rhamnose residues in A-PS preparations, but none have resolved the structural units described herein [18,21].

[0164] Example 2: Generation and immunological evaluation of glycoconjugates (BSA-OS2 and CRM-OS2) generated with isolated A-band epitope (OS2) Three mAbs specific for the A-PS terminus have been identified, and preliminary epitope mapping studies suggest that they recognize different regions within this terminus unit. These mAbs, and to a lesser extent, polyclonal sera raised against the A-PS terminus, were able to promote killing of P. aeruginosa strains in SBA and OPA experiments. Previous studies by Makarenko et al. using a synthetic glycoconjugate vaccine based on neutral rhamnan were unable to produce any recognizable antibodies that alone promoted killing of P. aeruginosa strains

[25] . In addition, studies by Lam's laboratory observed that the mAb N1F10 specific for neutral rhamnan was able to recognize approximately 70% of P. aeruginosa strains

[26] , but more recent studies with N1F10 or its scFv derivatives did not show any functionality

[27] . It is likely that key regions of this conserved antigen have been overlooked in previous studies, and thus the examples described herein demonstrate that the methylated tip region of A-PS is indeed immunogenic and that the immune response can promote opsonophagocytic activity. One of the three mAbs described herein, 1B1, appears to recognize a highly conserved epitope, as it recognized the majority of P. aeruginosa strains tested, including commonly encountered pathogenic serotypes and recent clinical isolates. Two other mAbs described herein, 3B8 and 3C4, are more specific than the 1B1 mAb, and their recognition is restricted to the wt serotype 5 strains PAO1 BAA-47 and 5937. Studies with synthetic oligosaccharides based on terminally methylated pentasaccharides confirmed the broad specificity of mAb 1B1 when compared to the other mAbs described herein. Inhibition ELISA data with synthetic oligosaccharides revealed that mAb 1B1 can be efficiently inhibited by 3-O-methyl rhamnan trisaccharide. Given the broad cross-reactivity of mAb 1B1, which recognizes the majority of clinical isolates in our collection and the ATCC serotypes responsible for the majority of disease caused by P. aeruginosa, the synthetic oligosaccharides described herein appear to hold promise as vaccine antigens or as a means to generate broadly cross-reactive therapeutics.

[0165] The importance of the A-PS antigen remains a subject of some discussion in the field, but the finding that in CF lungs, serotype-specific O antigens are no longer established but A-PS is maintained suggests that the A-PS immunogenic chip structure may be a key epitope to target in the clinical niche.

[0166] Conjugates: BSA and CRM conjugates were prepared by standard chemical techniques of direct reductive amination as previously described

[22] . Briefly, oxidized A-band terminal epitope material (OS2) (4.5 mg and 2 mg) was reacted with CRM (2.3 mg) or BSA (1 mg), respectively, by mixing solutions of the two reactants in water, leaving at RT, and then lyophilizing. The lyophilized material was dissolved in 10 mL of 100 mM NaPO4, then added NaCNBH3 (5 mg mL -1 ) was added and the resulting solution was left at room temperature (RT) for 16 h. The conjugate was purified with 10 mM sodium citrate in PBS on a 30 KDa molecular weight cutoff spin column, the supernatant protein quantified, filter sterilized, and frozen at -20°C. An aliquot was examined by MALDI-MS as previously described

[23] .

[0167] Generation of anti-P. aeruginosa A-band terminal epitope antibodies: To generate antibodies targeting the P. aeruginosa A-band terminal epitope, six Balb / C mice (6–8 weeks) and two New Zealand White rabbits (1.5–2 kg) were immunized with glycoconjugates of purified and oxidized terminal A-band molecules and CRM as a carrier protein. Immunizations were scheduled and adjuvanted as previously described

[22] .

[0168] mAb generation: Mice immunized against polyclonal sera were screened for recognition of A-band PS and mice showing the highest titers were selected for fusion after a final iv injection. All procedures for fusion of splenocytes harvested from immunized mice, hybridoma selection, and expansion and purification of high-density mAb supernatants were performed as previously described

[24] .

[0169] Characterization of anti-Pseudomonas aeruginosa band A terminal epitope mAb Antibody variable region sequencing: The DNA sequences encoding the rearranged variable heavy (VH) and variable light (VL) domains of the three generated mAbs (1B1, 3C4, and 3B8) were determined by Illumina MiSeq amplicon sequencing as previously described.

[24] The amino acid sequences of the VH and VL domains of the three mAbs are presented in Table 4.

[0170] [Table 4] TIFF2025509292000017.tif199149 TIFF2025509292000018.tif62149

[0171] ELISA: Binding of anti-P. aeruginosa band A terminal epitope mAb and polyclonal sera to BSA conjugates, purified LPS, and whole cells was assessed by ELISA as previously described

[24] .

[0172] mAb competitive ELISA: Wells of Nunc Maxisorp EIA plates were coated with 1 μg P. aeruginosa LPS in PBS overnight at 4° C. and then brought to RT before use. Plates were blocked with 1% BSA-PBS for 1 h at RT, and then wells were washed with PBS-T. One mAb (1B1 or 3B8) was titrated and added to the plate for 1 h at RT. After washing with PBS-T, the second mAb (3C4) was added at a pre-determined concentration and allowed to incubate for 1 h at RT. After washing with PBS-T, AP-labeled goat anti-mouse IgG2b (SouthernBiotech) specific for mAb 3C4 was added at 1:250 dilution in 1% BSA-PBS for 1 h at RT. Plates were then washed with PBS-T and developed with Phosphatase Substrate System (Kirkegaard and Perry Laboratories). After 60 min, absorbance at 405 nm was measured using a microtiter plate reader.

[0173] mAb Inhibition ELISA: Wells of Nunc Maxisorp EIA plates were coated with either 1 μg P. aeruginosa LPS in PBS overnight at 4° C. or with P. aeruginosa killed whole cells in dH2O overnight in a desiccator and then brought to RT before use. Plates were blocked with 1% BSA-PBS for 1 h at RT and then wells were washed with PBS-T. Inhibition was set up during the blocking step. Either LPS or synthetic oligosaccharides were added to the tubes at either 1 mg / ml or 3 mg / ml, then serial dilutions with 1% BSA-PBS were performed, after which an equal amount of mAb (1B1, 3B8 or 3C4) was added to 1% BSA-PBS at a constant concentration of 10 μg / ml and the mixture was incubated for 1 h at RT. After washing the EIA plates with PBS-T, 100 μl from the mixed tube was added to the plate and the LPS-coated plates were allowed to incubate for 1 h at RT or the killed whole cell-coated plates for 3 h at RT. After washing with PBS-T, the appropriate AP-conjugated goat anti-mouse Ig (SouthernBiotech) specific for the mAb used was added at 1:250 dilution in 1% BSA-PBS for 1 h at RT. Plates were then washed with PBS-T and developed with Phosphatase Substrate System (Kirkegaard and Perry Laboratories). After 60 min, absorbance at 405 nm was measured using a microtiter plate reader.

[0174] Serum bactericidal assays: The ability of polyclonal sera and mAbs to promote bactericidal injury of selected P. aeruginosa strains was determined as previously described

[22] .

[0175]

[0176] Opsonophagocytosis assay: The ability of polyclonal sera and mAbs to promote opsonophagocytic killing of selected P. aeruginosa strains was determined as previously described

[23] .

[0177] Surface Plasmon Resonance (SPR): SPR binding assays were performed at 25°C in HBS-EP running buffer (Cytiva LifeSciences, Mississauga, Canada) on a Biacore T200 instrument essentially as previously described

[24] . Briefly, approximately 13,000 RU of IgM (PA 1B1 and control Fn 4F1) were amine-coupled to a CM7S sensor chip in 10 mM acetate buffer, pH 4.0 (Cytiva). Synthetic oligosaccharides were reconstituted to 25 mM in HBS-EP and serial dilutions were prepared and injected across the IgM surface. Contact times were 60 s and dissociation times were 120 s (mono-, di-) or 180 s (tri-, tetra-, penta-). The following concentration ranges were injected: mono- (1 mM to 62.5 μM), di- (200 μM to 12.5 μM), and tri-, tetra-, and penta- (10 μM to 625 nM). Mcat lgt2 / 4 oligosaccharide from Moraxella catarrhalis acted as a control and was injected at 10 μM. Surfaces were regenerated by extensive washing with HBS-EP. Reference flow cell subtracted sensorgrams were fitted to a 1:1 binding model to determine binding kinetics and affinity or affinities were determined by steady state analysis.

[0178] Example 2: Results Conjugation: The reducing end erythritol of OS1 was efficiently targeted by periodate oxidation to generate a reactive aldehyde group for conjugation. The oxidized oligosaccharide OS2 (Figure 7) was conjugated to the carrier proteins BSA and CRM197 by direct reductive amination, and MALDI-MS analysis exemplified the attachment of ~13 and ~10 kDa carbohydrate conjugates (10–13 glycans / protein), respectively, to the resulting glycoconjugates (Figures 8A–8D).

[0179] Immunization: Six mice and two rabbits were subjected to the prime-one-boost-two-times immunization strategy with CRM glycoconjugates as described in the experiment. ELISA with terminal bleed sera highlighted that both animals seroconverted to the oligosaccharide conjugates, as evidenced by titrations to BSA conjugates and LPS (Figures 9A and 9B). Mice showed stronger titers to the BSA conjugates than to LPS, whereas rabbits showed broadly similar titration curves to both antigens.

[0180] Mab generation: Mice with the best titers against PAO1 LPS (NRCC6667) after glycoconjugate immunization were selected for mAb generation, and their spleens were fused to bone marrow cell lines as described in the experimental section. Three mAbs were obtained: 3C4 (IgG2b), 3B8 (IgM), and 1B1 (IgM). The heavy and light chain variable regions of these mAbs were sequenced. Their amino acid sequences are shown in Table 4. Each mAb was able to recognize the homologous PAO1(wzy::Gm) LPS and, in the case of mAb 1B1, P. aeruginosa purified LPS from A-band locus mutants PAO1(wzy::Gm)(Δpa5457) and PAO1(wzy::Gm)(Δpa5458) (Figure 10). Similarly, each mAb was able to recognize bactericidal whole cells of wt serotype 5 strains PAO1 BAA-47 and 5937, and again supporting the LPS ELISA data, mAb 1B1 was the only mAb that was cross-reactive to bactericidal whole cells of a subset that included A-band locus mutants PAO1(wzy::Gm)(Δpa5457), PAO1(wzy::Gm)(Δpa5458), and PAO1(wzy::Gm)(Δpa5459), as well as ATCC type strains corresponding to the most commonly encountered clinical serotypes (Figures S11A-S11C). This behavior was recapitulated when a set of clinical isolates was examined, with mAb 1B1 demonstrating broad cross-reactivity (Figure S12).

[0181] SBA and OPA: In the first attempt, we focused on demonstrating the ability of polyclonal rabbit sera to promote serum bactericidal injury of PAO1 wt P. aeruginosa strains. However, we observed limited evidence of injury by polyclonal rabbit sera. In the next attempt, we focused on opsonophagocytic assays (OPA) as literature reports have utilized OPA to demonstrate the potential of antisera against P. aeruginosa [8-10]. Similar to SBA, we obtained tentative evidence that rabbit sera could promote opsonophagocytic activity against PAO1 O antigen-deficient strains. However, when mAbs were examined, we observed high opsonophagocytic titers against PAO1 serotype 5 strains PAO1 BAA-47 and 5937 (Figures 13A and 13B).

[0182] mAb epitope mapping: Preliminary experiments were performed to determine whether the mAbs recognized identical, different, or overlapping epitopes on the 3-O-Me rhamnan. Competitive ELISA studies suggested that mAb 1B1 recognized a unique epitope compared to 3C4, whereas 3C4 and 3B8 recognized similar overlapping epitopes (Figures 14A and 14B), consistent with the 1B1 mAb having the ability to recognize a broader subset of strains compared to mAbs 3B8 and 3C4.

[0183] Inhibition ELISA with synthetic oligosaccharides: To further characterize the immunogenic epitopes recognized by the mAbs, synthetic oligosaccharides representing monosaccharides (D- and L-isomers), disaccharides (with and without linkers), trisaccharides, tetrasaccharides, and pentasaccharides of the 3-O-methyl D-rhamnan terminal units were prepared as described herein and examined in inhibition ELISA experiments. First, the experiments were validated using LPS molecules known to be recognized or not recognized by the three mAbs. Synthetic oligosaccharides were then used as inhibitors to reveal that trisaccharides, tetrasaccharides, and pentasaccharides were efficiently equivalent in inhibiting the binding of mAb 1B1 to target LPS and to killed whole cells (Figures 15A-15F). There was some evidence of inhibition by disaccharides and monosaccharides (D-isomers only), but not to the extent observed for trisaccharides and higher oligosaccharides. Inhibition by L-monosaccharides was completely abolished, confirming that the D-isomers are present in the correct configuration of the rhamnose sugar in 3-O-methyl rhamnan. None of the synthetic oligosaccharides were effective in inhibiting the binding of mAbs 3C4 or 3B8 to their LPS or bactericidal whole cell targets. These results are consistent with previous data and suggest that mAb 1B1 can recognize terminal 3-O-methyl rhamnan and that the trisaccharide may be sufficiently effective to mimic the native structure.

[0184] SPR Analysis: SPR was used to further define the minimal epitope recognized by 1B1 and to determine affinity for synthetic oligosaccharides (Figure 16 and Table 5). Synthetic carbohydrates ranging from monosaccharides to pentasaccharides were flowed over the high density surfaces of 1B1 IgM and an unrelated IgM, Fn 4F1. No observable binding was detected for monosaccharides up to 1 mM. Disaccharides showed specific binding to the 1B1 surface with fast association and dissociation rates, determining a steady state affinity of KD = 84 μM. Trisaccharides, tetrasaccharides, and pentasaccharides all bound specifically to 1B1 with affinities much higher than disaccharides (KD = 1.42, 0.66, and 0.55 μM, respectively). All three synthetic oligosaccharides had measurable kinetics, highlighted by slow dissociation rates not commonly observed in carbohydrate-antibody interactions. The observed Rmax values ​​were approximately 25% of the theoretical Rmax, indicating acceptable 1B1 IgM surface activity. When the unrelated oligosaccharide Mcat lgt2 / 4 (886 Da) from M. catarrhalis was injected at 10 μM, no observable binding was detected on either IgM surface. Taken together, these data suggest that although high 1B1 binding affinity was observed with tetrasaccharides and pentasaccharides, the trisaccharide has the potential to be the minimal antigen recognition domain that mimics the native structure.

[0185] [Table 5]

[0186] Example 3A - Synthesis of 3-O-methyl rhamnose oligosaccharides In this example, the synthesis of the target pentasaccharide is described (see schemes 1-3 in Figures 22-24). The synthesis of 3-OD-rhamnose and 3-OL-rhamnose and derivatization to the respective acetylated material with 2-octyl glycoside demonstrated the purity of each enantiomer produced by GC-MS. Large-scale synthesis of intermediate thioglycoside donor 16 and acceptor 17 yielded disaccharide 18di. After benzyl deprotection, new acceptor 19di was obtained, and further iterative glycosylation and hydrogenation reactions finally yielded 18 pentasaccharide. After global deprotection, the target pentasaccharide was synthesized with an overall yield of 1.0% from D-mannose. In addition, an aminoethyl linker was added to the disaccharide to construct a method for further conjugate vaccine production. The method described in this example illustrates that the synthesis can yield sufficient amounts of pure pentasaccharide and this glycan can be used to facilitate the production of P. aeruginosa glycoconjugate vaccines.

[0187] All chemicals were purchased from Aldrich, Fisher Scientific, AlphaAeser, or CombiBlocks. They were used without further purification. NMR spectra were recorded on a Varian spectrometer ( 1 H 500MHz, 13 C 125MHz, 31 P 200 MHz) and reported together with the solvent residual signal (CDCl3, 1 7.26 ppm for H and 13 C: 77.1 ppm, CD3OD, 1 3.31 ppm for H and 13 C: 49.0 ppm, D2O, 1 4.79 ppm for H and 13 In C, external dioxane (67.2 ppm) and 31 (For P, 85% H3PO4 was used in a separate experiment prior to spectral measurement.) Compound assignments were made using standard 2D NMR experiments, e.g., HMBC, COSY, HSQC, and 13The compound was confirmed using C NMR. The compound was purified using a CombiFlash® RF system and a RediSep® RF silica column. MS data was recorded on a Waters SQ2 and HRMS data was recorded on a Waters Ultima using Agilent LC / MS CalibrantMix as an internal standard.

[0188] 1,4-O-Dibenzyl-2,3-O-isopropylidene-α-D-rhamnopyranoside (4) 1-O-benzyl-2,3-O-isopropylidene-α-D-rhamnopyranoside (9.70 g, 32.9 mmol) was dissolved in anhydrous DMF (200 mL) and the solution was cooled to 0° C. Sodium hydride 60% in mineral oil (1.97 g, 49.3 mmol) was added in small portions and the solution was stirred at 0° C. for 30 min under nitrogen gas. Benzyl bromide (4.70 mL, 39.5 mmol) was then added dropwise and the reaction was stirred for 1 h after which it had reached completion as determined by TLC. Upon completion, the reaction was quenched with Et3N (8 mL) and poured into a cooled solution of saturated NH4Cl (500 mL). The product rapidly precipitated out of solution and was filtered, washed with water and dried under high vacuum overnight to yield product 4 (12.6 g, 32.9 mmol, 100%) as a beige solid. Rf = 0.75, (EtOAc / Hexane, 2 / 8) [α] D 25 23.45 (c 0.22, CHCl3) 1 HNMR (500 MHz, CDCl3): δ 7.38-7.26 (m, 10H, 2x Bn), 5.05 (s, 1H, H1α), 4.89 (d, 1H, J A,B = 11.6 Hz, CHA, OBn), 4.69 (d, 1H, J A,B = 11.8 Hz, CHA, OBn), 4.64(d, 1H, J B,A = 11.6 Hz, CHB, OBn), 4.51 (d, 1H, J B,A =11.8 Hz, CHB, OBn), 4.29 (dd, 1H, J 3,4= 6.2 Hz, J 3,2 =6.2 Hz, H3), 4.19 (d, 1H, J 2,3 = 5.8 Hz, H2), 3.80-3.73 (m, 1H, H5),3.24 (dd, 1H, J 4,3 = 7.2 Hz, J 4,5 = 9.9 Hz, H4), 1.51,1.36 (2 xs, 3H, CH3), 1.29 (d, 3H, J 6,5 = 6.3 Hz, H6) 13 CNMR (125 MHz, CDCl3): δ 138.4, 137.2, 128.6(x2), 128.4 (x2), 128.3 (x2), 128.1 (x2), 128.0, 127.7 (Bn), 109.3 (C(CH3)2),96.3 (C1), 81.3 (C4), 78.7 (C3), 76.2 (C2), 73.1, 69.1 (2 x CH2(Bn), 64.8 (C5), 28.1, 26.4 (2 x CH3), 17.9 (C6) J C1, H1 =174 Hz, HRMS: m / z C 23 H 28 NaO5[M+Na] + Calculated value: 407.1834; measured value: 407.1828.

[0189] 1,4-O-Dibenzyl-α-D-rhamnopyranoside (5) Compound 4 (0.46 g, 1.21 mmol) was dissolved in a solution of 80% (v / v) AcOH (8 mL) and the solution was heated at 60° C. for 4 h. The solution was then evaporated under reduced pressure and the crude product was crystallized from CHCl / Hexane to give 5 (395 mg, 1.15 mmol, 95%) as white crystals. Rf = 0.6 (EtOAc / Hexane, 7 / 3) [α] D 25 1.49 (c 0.09, CHCl3) 1HNMR (500 MHz, CDCl3): δ 7.36-7.26 (m, 10H,2x Bn), 4.86 (s, 1H, H1), 4.78-4.73 (m, 2H, CHA, CHB, OBn), 4.69 (d, 1H, J A,B = 11.9 Hz, CHA, OBn), 4.51 (d, 1H, J B,A = 11.9 Hz, CHB, OBn), 3.99-3.94 (m, 2H, H2, H3), 3.83-3.76 (m, 1H, H5), 3.37 (dd, 1H, J 4,3 = J 4,5 = 9.1 Hz, H4), 2.26 (d, 1H, J OH,2 = 3.6Hz, OH), 2.24 (d, 1H, J OH,3 = 4.8 Hz, OH), 1.36 (d, 3H, J6,5= 6.3 Hz, H6). 13 C NMR (125 MHz, CDCl3): δ 138.3, 137.3, 128.7 (x2), 128.6 (x2), 128.1, 128.1 (x2), 128.0(x2), 128.0, (Bn), 98.6 (C1), 81.8 (C4), 75.2 (CH2, Bn), 71.6 (C3),71.3 (C2), 69.2 (CH2, Bn), 67.5 (C5), 18.1 (C6) J C1, H1 =167 Hz LRMS m / z C 20 H 24 NaO5[M+Na] + Calculated value, 367.4; Measured value, 367.3

[0190] 1,4-ジ-O-ベンジル-3-O-メチル-α-D-ラムノピラノシド(6) Compound 5 (25.1 g, 72.9 mmol) was coevaporated with toluene three times and dried under high vacuum overnight. 5 was then dissolved in anhydrous toluene (500 mL), purged with nitrogen gas, and dibutyltin(IV) oxide (21.8 g, 87.5 mmol) was added. The reaction mixture was stirred at reflux for 16 h until cooled to RT and evaporated under vacuum. The crude mixture was further dried under high vacuum for 5 h until redissolved in 500 mL anhydrous DMF. The mixture was purged with nitrogen gas, and then cesium fluoride (16.5 g, 109 mmol) and iodomethane (51.7 mL, 830.4 mmol) were added. The reaction was stirred at 40° C. under nitrogen gas for 16 h, after which it reached completion as determined by TLC. The reaction mixture was cooled to RT, diluted with EtOAc (300 mL), and washed with water (5×60 mL), saturated NaHCO3 (1×60 mL), and brine (1×60 mL). The combined organic layers were then dried over Na2SO4, evaporated, and purified by flash chromatography (eluent: EtOAc / hexanes) to give 6 as a pale oil (23.7 g, 66.3 mmol, 91%-2 steps). Rf = 0.35 (EtOAc / hexanes, 7 / 3) [α] D 25 58.3 (c 0.43, CHCl3) 1 HNMR (500 MHz, CDCl3): δ 7.37-7.26 (m, 10H, 2x Bn), 4.91 (d, 1H, J 1,2 = 1.2 Hz, H1), 4.86 (d, 1H, J A,B = 11.0 Hz, CHA, OBn), 4.71 (d, 1H, J A,B = 11.8 Hz, CHA, OBn), 4.64(d, 1H, J B,A = 11.6 Hz, CHB, OBn), 4.47 (d, 1H, J B,A =11.0 Hz, CHB, OBn), 4.08 (dd, 1H, J 2,1 = 1.7 Hz, J 2,3 =3.3 Hz, H2), 3.83-3.73 (m, 1H, H5), 3.61 (dd, 1H, J 3,2 = 3.4 Hz, J3,4 = 9.1 Hz, H3), 3.49 (s, 3H, OMe), 3.39 (dd, 1H, J 4,3 = 9.4 Hz, J 4,5 = 9.4 Hz, H4), 2.43 (d, 1H, J OH,3 = 2.0 Hz, OH), 1.31 (d, 3H, J 6,5 = 6.3 Hz, H6). 13 C NMR (125 MHz, CDCl3): δ 138.6, 137.3, 128.5 (x2), 128.5 (x2), 128.1 (x2), 128.1 (x2),128.0, 127.8 (Bn), 98.3 (C1), 81.8 (C3), 80.0 (C4), 75.4, 69.2 (2 x CH2(Bn)), 68.0 (C2), 67.4 (C5), 57.5 (OCH3), 18.0 (C6) J C1, H1 =174 Hz HRMS: m / z C 21 H 26 NaO5[M+Na] + Calculated value: 381.1672; measured value: 381.1668.

[0191] 3-O-Methyl-D-rhamnose (D-monosaccharide) 1-O-Benzyl-3-O-methyl-α-D-rhamnopyranoside (43.7 mg, 162.9 μmol) was dissolved in a 16:10 (V:V) solution of 4 M HCl and MeCN (10 mL) and the solution was heated at 80° C. for 16 h. The solution was then evaporated and purified by flash chromatography (eluent: MeOH / CHCl, 1 / 5) to give the monosaccharide as a white solid (18.9 mg, 106.1 μmol, 65%). Rf = 0.30, (EtOAc), 1 H NMR (600 MHz, CD3OD): δ 5.18 (d, 0.7H, J 1α , 2α = 1.6 Hz, H1α), 4.74 (d, 0.3H, J 1β , 2β = 1.0 Hz, H1β), 4.07 (dd, 0.7H, J 2β,1β= 0.7 Hz, J.S 2β,3β = 2.9 Hz, H2β), 4.03 (dd, 0.7H, J 2α,1α = 0.9 Hz, J.S 2α,3α = 1.9 Hz, H2α), 3.89–3.83 (m, 0.7H, H5α), 3.50–3.48 (2xs, 3H, OCH3α / β), 3.46 (dd, 0.7H, J 4α,3α = 3.0 Hz, J 4α,5α = 9.4 Hz, H4α), 3.43–3.39 (m, 1H, H3α and H4β), 3.42–3.35 (m, 0.3H, H5β), 3.15 (dd, 1H, J). 3β,2β = 3.0 Hz, J 3β,4β = 9.2 Hz, H3β), 1.34–1.25 (m, 3H, H6α / H6β). 13 C NMR (151 MHz, CD3OD):δ 95.7(C1α), 95.4(C1β), 84.5(C3β), 81.8(C3α), 73.0(C4α), 72.4(C4β), 71.1(C5β), 69.2(C5α), 69.0(C2β), 68.8(C2α), 57.3(OCH3α), 57.2(OCH3β), 18.1(C6α), 18.1(C6β). J C1, H1α = 172 Hz, J C1,H1β = 161 Hz, HRMS: m / z C7H 14 NaO5[M+Na] + of Inventory, 201.0739; Documentation, 201.0734.NMR.

[0192] 3-O-linker-L-linker (L-sink) 1,2-Di-O-acetyl-3-O-methyl-L-rhamnopyranoside S9 (100 mg, 381 μmol) was dissolved in 10 mL of a solution of MeOH / HO / EtN (7 / 2 / 1) and the reaction was stirred at RT for 16 h. The solution was then evaporated under reduced pressure and filtered through Dowex® Na+ and then through a Sephadex® G-12 column. The fractions containing the product were collected and evaporated to give 13 as an α / β mixture (8 / 5) as a clear oil (37.5 mg, 210 μmol, 55%). 1 H NMR (600 MHz, CD3OD): δ 5.06 (d, 0.8H, J 1α , 2α = 1.7 Hz, H1α), 4.87 (d, 0.2H, J 1β , 2β = 1.7 Hz, H1β), 4.05 (d, 0.2H, J 2β,1β = 0.9 Hz, J 2β,3β = 3.1 Hz, H2β), 4.01 (dd, 0.8H, J 2α,1α = 1.8Hz, J 2α,3α = 2.9 Hz, H2α), 3.83 (m, 0.8H, H5α), 3.48 (s, 0.6H, OCH3β ), 3.48 (s, 2.4H, OCH3α ), 3.44(dd, 0.8H, , J 4α,3α = J 4α,5α = 9.6 Hz, H4α), 3.41-3.36 (m, 1H, H3α, H4β), 3.29 (m, 0.2H, H3β), 1.30 (d, 0.6H, J 6β,5β = 6.1 Hz ), 1.25 (d, 2.4H, J 6α,5α = 6.1 Hz, H6α). 13C NMR (151 MHz, CD3OD): δ 95.7 (C1α), 95.3 (C1β), 84.5 (C3β), 81.8 (C3α), 73.0 (C4α), 72.4 (C4β), 73.3 (C5β), 69.2 (C5α), 69.0 (C2β), 68.8 (C2α), 57.3 (OCH3α), 57.2 (OCH3β), 18.1 (C6α), 18.1 (C6β). J C1, H1α = 172 Hz, J C1,H1β = 162 Hz. HRMS: m / z C7H 14 NaO5[M+Na] + Calculated, 201.0733; Found, 201.0735. The NMR spectrum is shown in Figure 5A.

[0193] 4-O-Benzyl-3-O-methyl-D-rhamnopyranose (14) Compound 6 (1.54 g, 4.30 mmol) was dissolved in a 16:10 solution of 4 M HCl and MeCN (219 mL) and the solution was heated at 80° C. for 3 h. TLC analysis indicated that the reaction had reached completion after 3 h. The solution was then evaporated and purified by flash chromatography (eluent: MeOH / CH2Cl2, 1 / 5) to give compound 14 as an α / β mixture (6 / 5) as a clear oil (0.75 g, 2.80 mmol, 65%). Rf = 0.25 (EtOAc / Hexane, 1 / 1). 1 HNMR (500 MHz, CDCl3): δ 7.36-7.27 (m, 5H,Bn), 5.25 (s, 1H, H1α), 4.85 (d, 1H, J A,B =11.1 Hz, CHA, OBn), 4.72 (s, 1H, H1β), 4.63 (d, 1H, J B,A = 10.9 Hz, CHB, OBn), 4.10-4.08 (m, 2H, H2α, H2β), 3.99-3.92 (m, 1H, H5α), 3.62 (dd, 1H, J3α, 4α= 9.1 Hz, 3.3 Hz,H3α), 3.52, 3.50(2xs,3H,OCH3α / β), 3.38(dd,1H,J 4α,3α = 9.4 Hz, J.S 4α,5α = 9.4 Hz, H4α), 3.37–3.31 (m, 3H, H3β, H4β, H5β), 2.56 (br. s, 1H, OH), 2.46 (br. s,1H, OH), 1.33 (d, 3H, J). 6β,5β = 5.7 Hz, H6β), 1.29 (d, 3H, J 6α,5α = 5.7 Hz, H6α). 13 C NMR (125 MHz, CDCl3): δ 128.5, 128.4, 128.3, 128.0, 127.9, 127.8, 127.7 (Bn), 93.9 (C1α), 93.8 (C1β), 83.8 (C3β), 81.3 (C3α), 79.8 (C4α), 79.3(C4β), 75.2(CH2,Bn),71.1(C5β), 68.3(C2α), 68.0(C2β), 67.3(C5α), 57.5(OCH3α), 57.4(OCH3β),18.0(CH3,C6α), 17.9(CH3,C6β).HRMS m / z C 14 H 20 NaO5[M+Na] + of Construction, 291,1203; New York, 292.1205.

[0194] p-link2-link-4-O-link-3-O-link-1-link-D-link(16) Compound 15 (925 mg, 2.63 mmol) was dissolved in anhydrous CHCl (20 mL) and the solution was cooled to 0 °C under nitrogen gas. Thiocresol (456 mg, 3.67 mmol) was then added followed by boron trifluoride diethyl etherate (452.9 μL, 3.67 mmol). The reaction was stirred at 0 °C for 3 h until it reached completion. The solution was then diluted with CHCl (20 mL) and washed with water (2 × 20 mL) and saturated NaHCO solution (1 × 20 mL). The combined organic layers were then dried over NaSO, evaporated and purified by flash chromatography (eluent: EtOAc / Hexane) to give compound 16 as an α / β mixture (5 / 2) as a brown oil (880 mg, 2.11 mmol, 80%). Rf = 0.6 (EtOAc / Hexane, 3 / 7) [α] D 25 23.1 (c 2.1, CHCl3) 1 HNMR (500 MHz, CDCl3): δ 7.43-7.28 (m, 7H, Bn / Stol), 7.20-7.10 (m, 2H, Stol), 5.71 (d, 0.5H, J 2β,3β = 2.5 Hz, H2β), 5.56 (dd, 0.5H, J 2α,3α = 1.6 Hz, 3.2 Hz, H2α), 5.35 (d, 0.5H, J 1α,2α = 1.3 Hz, H1α), 4.92 (d, 0.5H, J Aα,Bα = 11.0 Hz, CHAα, OBn), 4.87 (d, 0.5H, J Aβ , Bβ = 11.0 Hz, CHAβ, OBn), 4.75 (s, 0.5H, H1β), 4.64 (dx 2,1H, J Bα,Aα及びBβ,Aβ = 10.7 Hz, CHBα and CHBβ, OBn), 4.27-4.20 (m, 0.5H, H5α), 3.65 (dd, 1H, J 3α,4α = 9.3 Hz, J 3α,2α= 3.3 Hz, H3α), 3.47 (s, 1.5H, OCH3α), 3.45 (s, 1.5H, OCH3β), 3.45 - 3.42 (m, 0.5H, H4α), 3.40 - 3.34 (m, 1.5H, H3β, H4β, H5β), 2.34 (s, 1.5H, CH3, STol β), 2.33 (s, 1.5H, CH3, STol α), 2.23 (s, 1.5H, Ac β), 2.15 (s, 1.5H, Ac α), 1.40 (d, 1.5H, J 6β,5β = 5.5 Hz, H6β), 1.35 (d, 1.5H, J 6α , 5α = 6.2 Hz, H6α). 13 C NMR (125 MHz, CDCl3): δ 170.5 (COβ), 170.3 (COα), 138.6, 138.5, 138.1, 138.0, 132.3 (x2), 132.3 (x2), 130.2, 130.2, 129.9 (x2), 129.9, 129.1, 128.5 (x2), 128.5, 128.3, 128.1, 128.0 (x2), 127.8, 127.8, 125.4 (Ar), 86.5 (C1α), 85.8 (C1β), 84.0 (C3β), 80.5 (C3α), 80.4 (C4α), 79.3 (C4β), 76.1 (C5β), 75.4 (CH2, Bn α, Bn β), 70.4 (C2α), 70.0 (C2β), 68.9 (C5α), 57.8 (OCH3β), 57.7 (OCH3α), 21.2 (CH3, Stol β), 21.2 (CH3, Stol α), 21.1 (Ac α), 20.9 (Ac β), 18.3 (C6β), 17.9 (C6α). J C1, H1α = 169 Hz, J C1,H1β = 154 Hz, ESI - MS: m / z C 23 H 28 NaO5S[M + Na] + Calculated value of, 439.1550; Measured value, 439.1545.

[0195] 1,2-Diacetyl-3-O-methyl-α-D-rhamnopyranoside (17) Compound 15 (450 mg, 1.28 mmol) was dissolved in 10.0 mL of MeOH and the solution was charged with palladium hydroxide on carbon (40 mg, 0.28 mmol). The solution was bubbled with a balloon of hydrogen gas for 5 min and stirred under an atmosphere of hydrogen gas for 3 h until completion was reached. The solution was then filtered through Celite and evaporated to give pure 17 as a clear oil (323 mg, 1.23 mmol, 96%). Rf = 0.25 (EtOAc / Hexane, 1 / 1) [α] D 25 -21.2 (c 3.4, CHCl3) 1 HNMR (500 MHz, CDCl3): δ = 5.96 (s, 1H, H1),5.24 (d, 1H, J 2,1 = 2.0 Hz, H2), 3.76-3.67 (m, 1H, H5), 3.48 (dd,1H, J 4,5 = 9.4 Hz, J 4,3 = 9.4 Hz, H4), 3.43 (dd, 1H, J 3,2 = 3.2 Hz, J 3,4 = 9.5 Hz, H3), 3.37 (s, 3H, OCH3),2.96 (br. s, 1H, OH), 2.08, 2.07 (2x s, 6H, CH3), 1.28 (d, 3H, J 6,5 = 6.3, H6) 13 C NMR (125 MHz, CDCl3): δ 169.9, 168.5 (2x C=O), 91.2 (C1), 79.3 (C3), 71.1 (C4), 70.4 (C5),66.4 (C2), 57.4 (OCH3), 20.9, 20.7 (2x Ac), 17.7 (C6).J C1, H1 =170 Hz, LRMS m / z C 11 H 18 NaO7[M+Na] + Calculated value, 285.1; measured value, 285.1.

[0196] 2-O-Acetyl-4-O-benzyl-3-O-methyl-α-D-rhamnopyranoside-(1→4)-1,2-O-diacetyl-3-O-methyl-α-D-rhamnopyranoside (18di) Compound 16 (615 mg, 1.48 mmol) and compound 17 (323 mg, 1.23 mmol) were coevaporated with toluene (5×10 mL) and dried under high vacuum overnight. Anhydrous CHCl (20 mL) was added followed by 0.6 g of activated powdered 3 Å molecular sieves. The reaction was then cooled to −78° C. under a nitrogen atmosphere and N-iodosuccinimide (406 mg, 1.80 mmol) was added followed by triflic acid (20 μL, 226 μmol). The reaction was stirred for 3 hours until completion was reached. The reaction was then filtered through a Buchner funnel, diluted with 20 mL of CHCl, and washed with 10% NaSO (2×20 mL) and saturated NaHCO solution (1×20 mL). The organic layer was then dried over Na2SO4, filtered, and purified by flash chromatography (eluent: EtOAc / Hexanes) to give compound 18di as a clear oil (363 mg, 655 μmol, 53%). Rf = 0.55 (EtOAc / Hexanes, 1 / 1) [α] D 25 183 (c 18.7, CHCl3) 1 HNMR (500 MHz, CDCl3) δ 7.40-7.16 (m, 5H,Bn), 6.02 (d, 1H, J 1,2 = 1.7 Hz, H1), 5.39 (dd, 1H, J 2',3' =1.9 Hz, 3.0 Hz, H2'), 5.31 (dd, 1H, J 2,1 = 1.7, 1.7 Hz, H2), 5.17(d, 1H, J 1',2' = 1.7 Hz, H1'), 4.92 (d, 1H, J A,B = 10.9Hz, CHA, OBn), 4.65 (d, 1H, J B,A= 10.9 Hz, CHB, OBn), 3.88-3.81 (m,1H, H5), 3.79-3.72 (m, 1H, H5'), 3.64-3.59 (m, 3H, H3, H3', H4), 3.48, 3.45 (2x s, 6H, OCH3), 3.40 (dd, 1H, J 4',5' = J 4',3' =9.5 Hz, H4'), 2.17, 2.16, 2.15 (3 xs, 9H, Ac), 1.35 (d, 3H, J 6',5' =6.2 Hz, H6'), 1.34 (d, 3H, J 6,5 = 6.1 Hz, H6). 13 C NMR(125 MHz, CDCl3): δ 170.2, 170.0, 168.6 (3 xC=O), 138.5, 128.5 (x2), 128.1 (x2), 127.8 (Bn), 99.5 (C1'), 91.1 (C1), 80.0(C4, C3'), 79.9 (C4'), 77.9 (C3), 75.5 (CH2, Bn), 69.3 (C5'), 68.7(C5), 68.6 (C2'), 66.9 (C2), 57.6, 57.6 (2x OCH3), 21.2, 21.0, 20.9(3 x Ac), 18.3 (C6), 18.0 (C6'). J C1, H1 = 173 Hz, J C1', H1' =179 Hz LRMS m / z C 27 H 38 NaO 12 [M+Na] + Calculated value, 577.2; Measured value, 577.9.

[0197] 2-O-アセチル-3-O-メチル-α-D-ラムノピラノシド-(1→4)-1 ,2-O-ジアセチル-3-O-メチル-α-D-ラムノピラノシド(19di) The same procedure as for 17. Compound 18di (363 mg, 655 μmol), palladium hydroxide on carbon (40 mg, 285 μmol), MeOH (10 mL). Compound 19di (273 mg, 588 μmol, 92%) was isolated as a clear oil. Rf = 0.2 (EtOAc / hexane, 1 / 1) [α] D 25 44 (c 4.3, CHCl3) 1 1H NMR (500 MHz, CDCl3) δ 5.93 (d, 1H, J 1,2 = 1.6 Hz, H1), 5.31 (dd, 1H, J 2,1 = 1.9 Hz, J 2,3 = 2.8 Hz, H2'), 5.22 (m, 1H, H2), 5.09 (d, 1H, J 1',2' = 1.7 Hz, H1'), 3.76 - 3.66 (m, 2H, H5, H5'), 3.54 - 3.51 (m, 2H, H3, H4), 3.44 (dd, 1H, J 4',5' = 9.4 Hz, J 4',3' = 9.4 Hz, H4'), 3.35, 3.34 (2 x s, 6H, OCH3), 3.34 - 3.29 (m, 1H, H3'), 2.88 (br. s, 1H, OH), 2.08, 2.06, 2.05 (3 x s, 9H, Ac), 1.29 - 1.25 (m, 6H, H6', H6). 13 13C NMR (125 MHz, CDCl3): δ 170.1, 170.0, 168.5 (3 x C=O), 99.7 (C1'), 91.0 (C1), 79.9 (C4), 79.4 (C3'), 77.5 (C3), 71.4 (C4'), 69.2 (C5'), 69.0 (C5), 67.4 (C2), 66.8 (C2'), 57.5, 57.2 (OCH3), 21.0, 20.9, 20.8 (3 x Ac), 18.2 (C6), 17.5 (C6'). J C1,H1 = 173 Hz, J C1',H1' = 168 Hz LRMS m / z C 20 H 32 NaO 12[M+Na] + Calculated value, 487.2; measured value, 486.9.

[0198] 2-O-acetyl-4-O-benzyl-3-O-methyl-α-D-rhamnopyranoside-(1→4)-2-O-acetyl-3-O-methyl-α-D-rhamnopyranoside-(1→4)-1,2-O-diacetyl-3-O-methyl-α-D-rhamnopyranoside (18 tri) Compound 16 (243 mg, 584 μmol) and compound 19di (226 mg, 487 μmol) were coevaporated with toluene (5×10 mL) and dried under high vacuum overnight. Anhydrous CHCl (10 mL) was added followed by 0.3 g of activated powder 3 Å molecular sieves. The reaction was then cooled to −78° C. under a nitrogen atmosphere and N-iodosuccinimide (161 mg, 715 μmol) was added followed by triflic acid (10 μL, 115 μmol). The reaction was stirred for 3 hours until completion was reached. The reaction was then filtered through a Buchner funnel, diluted with 20 mL of CHCl, and washed with 10% NaSO (2×10 mL) and saturated sodium bicarbonate solution (1×10 mL). The organic layer was then dried over Na2SO4, filtered, and purified by flash chromatography (eluent: EtOAc / Hexanes) to give compound 18 tri as a clear oil (254 mg, 336 μmol, 69%). Rf = 0.55 (EtOAc / Hexanes, 1 / 1), [α] D 25 157 (c15, CHCl3) 1 HNMR (500 MHz, CDCl3): δ 7.36-7.25 (m,5H, Bn), 5.98 (d, 1H, J 1,2 = 1.7 Hz, H1), 5.36-5.32 (m, 2H, H2',H2''), 5.28 (m, 1H, H2), 5.11 (m, 2H, H1', ​​H1''), 4.87 (d, 1H, J A,B = 10.9 Hz, CHA, OBn), 4.62 (d, 1H, J B,A= 10.9 Hz, CHB, OBn), 3.85 - 3.78 (m, 1H, H5''), 3.78 - 3.70 (m, 2H, H5', H5), 3.60 - 3.54 (m, 3H, H3', H3'', H4), 3.54 - 3.47 (m, 2H, H3, H4'), 3.43 (s, 3H, OCH3), 3.39 (s, 6H, OCH3x2), 3.36 (dd, 1H, J 4'',3'' = 9.6 Hz, H4''), 2.12 (s, 6H, Ac x 2), 2.11 (s, 3H, Ac), 2.08 (s, 3H, Ac), 1.31 (d, 3H, J 6'',5'' = 6.2 Hz, H6''), 1.30 (d, 3H, J 6,5 = 6.3 Hz, H6), 1.29 (d, 3H, J 6',5' = 6.3 Hz, H6'). 13 C NMR (125 MHz, CDCl3): δ 170.1, 170.0, 169.9, 168.4 (C=O), 138.5, 128.4 (x2), 128.0 (x2), 127.7 (Bn), 99.4 (C1'), 99.3 (C1''), 91.0 (C1), 79.9 (C4''), 79.9 (C3), 79.8 (C3'), 79.8 (C3''), 78.2 (C4'), 78.0 (C4), 75.3 (CH2, Bn), 69.1 (C5), 68.6 (C2'), 68.4 (C2''), 67.8 (C5'), 67.8 (C5''), 66.7 (C2), 57.5 (OCH3), 57.2 (OCH3), 21.1, 21.0, 20.9, 20.8 (Ac), 18.2 (C6''), 18.0 (C6'), 17.8 (C6) J C1,H1 = 179 Hz, J C1',H1' = 179 Hz, J C1'', H1'' = 179 Hz HRMS m / z C 36 H 52 NaO 17 [M + Na] + Calculated value of, 779.3102; Measured value, 779.3079.

[0199] 2-O-acetyl-3-O-methyl-α-D-rhamnopyranoside-(1→4)-2-O-acetyl-3-O-methyl-α-D-rhamnopyranoside-(1→4)-1,2-O-diacetyl-3-O-methyl-α-D-rhamnopyranoside (19 tri) Same procedure as 17. Compound 18 tri (228 mg, 301 μmol), palladium hydroxide on carbon (40 mg, 285 μmol), MeOH (10 mL). Compound 19 tri (195.5 mg, 293 μmol, 97%) was isolated as a clear oil. Rf = 0.25 (EtOAc / Hexane, 1 / 1) [α] D 25 118 (c 13.3, CHCl3) 1 HNMR (500 MHz, CDCl3): δ 5.98 (d, 1H, J 1,2 = 1.4 Hz, H1), 5.36-5.32 (m, 2H, H2', H2''), 5.2 (m, 1H, H2), 5.14 (d, 1H, J 1',2' = 0.9 Hz, H1'), 5.10 (d, 1H, J 1'',2'' = 1.2 Hz, H1''), 3.82-3.72 (m,3H, H5, H5', H5''), 3.58-3.52 (m, 3H, H3, H3', H4'), 3.52-3.47 (m, 2H, H3'',H4''), 3.40, 3.39, 3.38 (3x s, 3H, OCH3), 3.41-3.36 (m, 1H, H4),2.48 (br. s, 1H, OH), 2.13 (s, 3H, Ac), 2.11 (s, 3H, Ac), 2.09 (s, 3H, Ac), 2.08(s, 3H, Ac), 1.35-1.29 (m, 9H, H6, H6', H6''). 13C NMR (125 MHz,CDCl3): δ 170.1, 170.1, 170.0, 168.5 (4xC=O), 99.6 (C1'), 99.5 (C1''), 91.1 (C1), 80.0 (C3''), 79.9 (C3), 79.4 (C3'),78.1 (C4), 78.0 (C4'), 71.6 (C4''), 69.2 (C5), 68.9 (C5'), 67.9 (C5''), 67.8(C2''), 67.4 (C2'), 66.8 (C2), 57.6, 57.3, 57.2 (3x OCH3), 21.1(x2), 21.0, 20.9 (4x Ac), 18.3 (C6''), 18.1 (C6'), 17.6 (C6). J C1,H1 =172 Hz, J C1', H1' = 171 Hz, J C1'', H1'' = 171 Hz LRMS m / zC 29 H 46 NaO 17 [M+Na] + Calculated value, 689.3; measured value, 688.8.

[0200] 2-O-acetyl-4-O-benzyl-3-O-methyl-α-D-rhamnopyranoside-(1→4)-2-O-acetyl-3-O-methyl-α-D-rhamnopyranoside-(1→4)-2-O-acetyl-3-O-methyl-α-D-rhamnopyranoside-(1→4)-1,2-O-diacetyl-3-O-methyl-α-D-rhamnopyranoside(18tetra) Compound 16 (146 mg, 351 μmol) and compound 19 (195 mg, 293 μmol) were coevaporated with toluene (5×10 mL) and dried under high vacuum overnight. Anhydrous CHCl (10 mL) was added followed by 0.2 g of activated powder 3 Å molecular sieves. The reaction was then cooled to −78° C. under a nitrogen atmosphere and N-iodosuccinimide (96.5 mg, 429 μmol) was added followed by triflic acid (5 μL, 57.5 μmol). The reaction was stirred for 3 hours until completion was reached. The reaction was then filtered through a Buchner funnel, diluted with 10 mL of CHCl and washed with 10% NaSO (2×5 mL) and saturated NaHCO solution (1×5 mL). The organic layer was then dried over Na2SO4, filtered, and purified by flash chromatography (eluent: EtOAc / Hexanes) to give compound 18 tetra as a clear oil (149.7 mg, 156 μmol, 53%). Rf = 0.55 (EtOAc / Hexanes, 1 / 1) [α] D 25 54.7 (c 5.0, CHCl3) 1 HNMR (500 MHz, CDCl3): δ 7.37-7.26 (m, 5H,Bn), 5.99 (d, 1H, J 1,2 = 1.7 Hz, H1), 5.37-5.32 (m, 3H, H2', H2'',H2'''), 5.28 (m, 1H, H2), 5.11 (s, 3H, H1', ​​H1'', H1'''), 4.86 (d, 1H, J A,B = 10.9 Hz, CHA, OBn), 4.62 (d, 1H, J B,A= 10.9 Hz, CHB, OBn), 3.85 - 3.71 (m, 4H, H5, H5', H5'', H5'''), 3.61 - 3.53 (m, 4H, H3, H4, H4', H4''), 3.53 - 3.49 (m, 3H, H3', H3'', H3'''), 3.44, 3.40, 3.39, 3.38 (4 x s, 12H, OCH3), 3.37 - 3.31 (m, 1H, H4'''), 2.13, 2.12 (2 x s, 6H, Ac), 2.11, 2.08, 2.07 (3 x s, 9H, Ac), 1.34 (d, 3H, J 6,5 = 6.2 Hz, H6), 1.31 (d, 3H, J 6',5' = 6.3 Hz, H6'), 1.30 (d, 3H, J 6'',5'' = 6.3 Hz, H6''), 1.28 (d, 3H, J 6''',5''' = 6.3 Hz, H6 '''). 13 C NMR (125 MHz, CDCl3): δ 170.1, 170.0, 170.0, 169.9, 168.5 (C=O), 138.5, 128.4 (x2), 128.0 (x2), 127.8, (Bn), 99.5 (C1'''), 99.4 (C1''), 99.3 (C1'), 91.0 (C1), 80.0 (C3), 80.0 (C3'), 79.8 (C3'', C3''' and C4'''), 78.3 (C4), 78.1 (C4' and C4''), 75.4 (CH2, Bn), 69.1 (C5), 68.6 (C2'''), 68.4 (C5'''), 67.9 (C2''), 67.8 (C2'), 67.7 (C5'), 67.7 (C5''), 66.8 (C2), 57.5, 57.5, 57.3, 57.2 (4 x OCH3), 21.1, 21.0, 21.0, 21.0, 20.9 (5x Ac), 18.3 (C6), 18.1 (C6'), 18.1 (C6''), 18.0 (C6'''). J C1,H1 = 185 Hz, J C1',H1' = 179 Hz, J C1'',H1''=179 Hz , J C1''',H1''' = 179 Hz, HRMS m / z C 45 H 66 NaO 22 [M+Na] + Calculated value, 981.3943; measured value, 981.3910.

[0201] 2-O-acetyl-3-O-methyl-α-D-rhamnopyranoside-(1→4)-2-O-acetyl-3-O-methyl-α-D-rhamnopyranoside-(1→4)-2-O-acetyl-3-O-methyl-α-D-rhamnopyranoside-(1→4)-1,2-O-diacetyl-3-O-methyl-α-D-rhamnopyranoside(19tetra) Same procedure as 17. Compound 18 tetra (110 mg, 114.7 μmol), palladium hydroxide on carbon (20.0 mg, 143 μmol), MeOH (10 mL). Compound 19 tetra (95.6 mg, 110.0 μmol, 96%) was isolated as a white solid. Rf = 0.40 (EtOAc / Hexane, 1 / 1) [α] D 25 52.40 (c 4.78, CHCl3) 1 HNMR (600 MHz, CD3OD): δ 5.96 (d, 1H, J 1,2 = 1.6 Hz, H1), 5.41 (dd, 1H, J 2',1' = 3.0 Hz, J 2',3' = 5.6 Hz, H2'), 5.40 (dd, 1H, J 2'',1'' = 3.0 Hz, J 2'',3'' = 5.1 Hz, H2''), 5.37 (s, 1H, H2'''), 5.33 (dd, J 2,1 = 3 Hz, J 2,3 = 5.8 Hz, H2), 5.08 (m, 2H, H1', ​​H1'''), 5.07 (m, 1H, H1''), 3.88-3.80 (m, 3H,H5, H5', H5''), 3.80-3.73 (m, 1H, H5'''), 3.69 (dd, 1H, J 3,2 = 3.9Hz, J 3,4= 11.2 Hz, H3), 3.61-3.55 (m, 2H, H3', H3''), 3.55-3.45 (m,3H, H4, H4', H4''), 3.42-3.32 (m, 2H, H3''', H4'''), 3.41 (2 xs, 3H, OCH3),3.40 (2 xs, 3H, OCH3), 2.15, 2.13, 2.11, 2.11, 2.09 (5 xs, 15H, Ac),1.34-1.26 (m, 12H, H6, H6', H6'', H6'''). 13 C NMR (125 MHz, CD3OD / CDCl 3, 1 / 1): δ 171.0, 170.9, 170.9, 170.7, 169.3 (C=O), 99.9(C1'''), 99.8 (C1''), 99.8 (C1'), 91.4 (C1), 80.4 (C3''), 80.3 (C3'),80.2 (C3), 79.4 (C3'''), 78.9 (C4), 78.8 (C4'), 78.5 (C4''), 71.8 (C4'''), 69.8(C5'''), 69.5 (C5), 68.6 (C2'''), 68.5 (C2'), 68.4 (C2''), 68.2 (C5', C5''),67.3 J C1,H1 =179 Hz, J C1',H1' = 177 Hz, J C1'',H1'' =177 Hz , J C1''',H1''' = 176 Hz HRMS m / z C 38 H 60 NaO 22 [M+Na] + Calculated value, 891.3474; Measured value, 891.3429.

[0202] 2-O-acetyl-4-O-benzyl-3-O-methyl-α-D-rhamnopyranoside-(1→4)-2-O-acetyl-3-O-methyl-α-D-rhamnopyranoside-(1→4)-2-O-acetyl-3-O-methyl-α-D-rhamnopyranoside-(1→4)-2-O-acetyl-3-O-methyl-α-D-rhamnopyranoside-(1→4)-1,2-O-diacetyl-3-O-methyl-α-D-rhamnopyranoside(18penta) Compound 16 (37.4 mg, 89.9 μmol) and compound 19 tetra (48.8 mg, 56.2 μmol) were coevaporated with toluene (5×10 mL) and dried under high vacuum overnight. Anhydrous CHCl (5 mL) was added followed by 50 mg of activated powdered 3 Å molecular sieves. The reaction was then cooled to −78° C. under a nitrogen atmosphere and N-iodosuccinimide (24.7 mg, 109.8 μmol) was added followed by triflic acid (10 μL, 113 μmol). The reaction was stirred for 3 hours until completion was reached. The reaction was then filtered through a Buchner funnel, diluted with 10 mL of CHCl and washed with 10% NaSO (2×5 mL) and saturated NaHCO solution (1×5 mL). The organic layer was then dried over Na2SO4, filtered and purified by flash chromatography (eluent: EtOAc / Hexanes) to give compound 18 penta as a clear oil (35.6 mg, 30.7 μmol, 55%). Rf = 0.70 (EtOAc / Hexanes, 7 / 3) [α] D 25 22.26 (c 1.75, CHCl3) 1 HNMR (500 MHz, CDCl3): δ 7.36-7.26 (m, 5H,Bn), 5.98 (d, 1H, J 1,2 = 1.5 Hz, H1), 5.35-5.31 (m, 4H, H2',H2'', H2''', H2''''), 5.27 (s, 1H, H2), 5.10 (s, 4H, H1', ​​H1'', H1''', H1''''),4.86 (d, 1H, J A,B = 10.9 Hz, CHA, OBn), 4.61 (d, 1H, J B,A= 10.9 Hz, CHB, OBn), 3.84-3.70 (m, 5H, H5, H5', H5'', H5''', H5''''),3.60-3.51 (m, 5H, H3, H4, H4', H4'', H3''''), 3.51-3.46 (m, 4H, H3', H3'',H3''', H4'''), 3.43, 3.39 (2 x s, 3H, OCH3), 3.38 (s, 6H, OCH3x2),3.37 (s, 3H, OCH3), 3.37-3.31 (m, 1H, H4''''), 2.12, 2.12, 2.11,2.08 (4 x s, 12H, Ac), 2.07 (s, 6H, Ac x2), 1.36-1.26 (m, 15H, H6, H6', H6'',H6''', H6''''). 13 C NMR (125 MHz, CDCl3): δ 170.2, 170.1, 170.1, 170.0, 169.9, 168.5 (C=O), 138.5, 128.4 (x2),128.0 (x2), 127.8 (Bn), 99.5 (C1'''), 99.4 (C1''), 99.4 (C1'), 99.3(C1''''), 91.0 (C1), 80.0 (C3''''), 80.0 (C3), 79.9 (C3'), 79.9 (C3'', C3''',C4''''), 78.3 (C4), 78.3 (C4'), 78.2 (C4''), 78.1 (C4'''), 75.4 (CH2,Bn), 69.1 (C5), 68.7 (C2''''), 68.4 (C5''''), 67.9 (C2''), 67.9 (C2'''), 67.8 (C2'), 67.8 (C5'), 67.7 (C5'', C5'''), 66.8 (C2), 57.5, 57.5, 57.3, 57.3,57.2 (5 x OCH3), 21.1, 21.1, 21.0, 21.0, 21.0, 20.9 (6x Ac), 18.3(C6), 18.1 (C6'), 18.1 (C6''), 18.1 (C6'''), 17.9 (C6'''') J C1,H1 =174 Hz, J C1',H1'= 177 Hz, J C1'',H1'' =177 Hz , J C1''',H1''' = 177 Hz, J C1'''',H1'''' = 177 Hz HRMS m / z C 54 H 80 NaO 27 [M+Na] + Calculated value: 1183.4785; Measured value: 1180.4753.

[0203] 2-O-acetyl-3-O-methyl-α-D-rhamnopyranoside-(1→4)-2-O-acetyl-3-O-methyl-α-D-rhamnopyranoside-(1→4)-2-O-acetyl-3-O-methyl-α-D-rhamnopyranoside-(1→4)-2-O-acetyl-3-O-methyl-α-D-rhamnopyranoside-(1→4)-1,2-O-diacetyl-3-O-methyl-α-D-rhamnopyranoside(19penta) Same procedure as 17. Compound 18 penta (35.0 mg, 30.1 μmol), palladium hydroxide on carbon (15.0 mg, 105.0 μmol), MeOH (5 mL). Compound 19 penta (32.2 mg, 30.1 μmol, 100%) was isolated as a white solid. Rf = 0.45 (EtOAc / Hexane, 7 / 3) [α] D 25 19.6 (c 1.66, CD3OD) 1 H NMR (500 MHz, CD3OD): δ5.97 (d, 1H, J 1,2= 1.4 Hz, H1), 5.34-5.29 (m, 4H, H2', H2'',H2''', H2''''), 5.26 (s, 1H, H2), 5.12 (s, 1H, H1''''), 5.09 (s, 3H, H1', H1'',H1'''), 3.81-3.72 (m, 5H, H5, H5', H5'', H5''', H5''''), 3.57-3.35 (m, 4H, H3,H4', H4'', H4'''), 3.51-3.44 (m, 5H, H4, H4'''', H3', H3'', H3'''), 3.38 (2 xs, 3H, OCH3), 3.37 (s, 6H, OCH3), 3.36 (s, 3H, OCH3x2), 3.37-3.31 (m, 1H, H3''''), 2.12, 2.12, 2.10, 2.08, 2.07 (4 x s, 12H, Ac),2.06 (s, 6H, Ac x2), 1.35-1.25 (m, 15H, H6, H6', H6'', H6''', H6''''). 13CNMR (125 MHz, CD3OD): δ 170.1 (x2), 170.1,170.1, 170.0, 168.5 (C=O), 99.6 (C1''''), 99.6 (C1''), 99.5 (C1'), 99.4(C1'''), 91.1 (C1), 80.1 (C3''), 80.0 (C3'), 80.0 (C3), 79.9 (C3'''), 79.4(C3''''), 78.3 (C4'), 78.3 (C4''), 78.2 (C4'''), 78.1 (C4), 71.6 (C4''''), 69.2(C5), 68.9 (C5''''), 68.0 (C2'''), 68.0 (C2''), 67.9 (C2''''), 67.8 (C2'), 67.8(C5'), 67.7 (C5''), 67.5 (C5'''), 66.8 (C2), 57.6, 57.3, 57.3, 57.3, 57.2 (5 xOCH3), 21.1 LRMS m / z C 47 H 74 NaO 27 [M+Na] + Calculated value, 1093.4; measured value, 1093.8.

[0204] 3-O-methyl-α-D-rhamnopyranoside-(1→4)-3-O-methyl-D-rhamnopyranose (disaccharide) Compound 18di (18 mg, 38.7 μmol) was dissolved in MeOH (5 mL) and sodium metal (1 mg) was added to the reaction mixture. After 16 h, rexin-H + was added to the mixture until a pH of 5 was obtained. The mixture was then filtered and evaporated to give the disaccharide compound without further purification (13.1 mg, 38.7 μmol, 100% yield). Rf = 0.5 (MeOH / CH2Cl2, 1 / 5). 1 H NMR(600 MHz, CD3OD): δ 5.08 (d, 1H, J 1',2= 1.8 Hz, H1'), 4.99 (d, 0.8H, J 1α,2 = 1.7 Hz, H1α), 4.65 (d, 0.2H, J 1β,2 = 0.9 Hz, H1β), 4.04 (dd, 1H, J 2',1' = 1.9 Hz, J 2',3' = 3.1Hz, H2'), 3.99 (dd, 0.2H, J 2β,1β = 1.1 Hz, J 2β,3β 3.9 Hz, H2β), 3.96 (dd, 0.8H, J 2α,1α = 1.9 Hz, J 2α,3α = 2.9 Hz, H2α), 3.82 (m, 0.8H, H5α), 3.68 (m, 1H, H5'),3.51 (dd, 0.8H, J 4α,5α = 3.0 Hz, J 4α,3α = 9.3 Hz, H4α), 3.48 (m, 0.2H, H4β) 3.46 (dd, 0.8H, J 3α, 2α = 3.0 Hz, J 3α, 4α = 9.3 Hz, H3α), 3.42 - 3.40 (m, 4H, H4' and OCH3' ), 3.38 (s, 3H, OCH3), 3.31(m, 0.2H, H5β), 3.24 (dd, 1H, J 3',2' = 3.2Hz, J 3',4' = 9.4 Hz, H3'), 3.23 (m, 0.2H, H3β), 1.34 (d, 0.6H, J 6β,5β = 6.0 Hz, H6β) 1.24 (d, 2.4H, J 6α,5α = 6.3 Hz, H6α), 1.20 (d, 3H, J 6',5' = 6.4 Hz, H6'). 13C NMR(150 MHz, CD3OD): δ 103.2 (C1'), 95.5 (C1α), 95.4 (C1β), 85.5 (C3β), 83.0 (C3α), 82.0 (C3'), 79.8 (C4α), 79.2 (C4β), 72.6 (C4'), 72.0 (C5β), 70.5 (C5'), 68.5 (C2α), 68.4 (C2β and C2'), 67.8 (C5α), 57.3 (OCH3'),56.7 (OCH3α), 56.5 (OCH3β), 18.8 (C6α and C6β),17.9 (C6').J C1', H1'α = 173 Hz, J C1,H1α = 172 Hz, J C1, H1β = 165 Hz. The NMR spectrum is shown in Figure 17.

[0205] 3-O-methyl-α-D-rhamnopyranoside-(1→4)-3-O-methyl-α-D-rhamnopyranose (trisaccharide) Compound 19 trisaccharide (25 mg, 37.5 μmol) was dissolved in 10 mL of anhydrous MeOH and the solution was stirred under a nitrogen atmosphere. Sodium metal (10.0 mg, 0.44 mmol) was then added and the reaction was stirred before reaching completion as determined by TLC. The solution was then neutralized with lexan, filtered through Celite, and evaporated to give the trisaccharide compound as a clear solid (18.1 mg, 36.3 μmol, 97%). Rf = 0.45 (MeOH / CH2Cl2, 1 / 4) [α] D 25 16.754 (c 1.4, MeOH) 1 HNMR (600 MHz, CD3OD): δ 5.16 (m, 2H, H1' and H1''), 5.07 (d, 0.8H, J 1α,2 = 1.6 Hz, H1α), 4.65 (d, 0.2H, J 1β,2 = 0.6 Hz, H1β), 4.12 (dd, 1H, J 2',1' = 2.2 Hz, J 2',3' = 2.6Hz, H2'), 4.11 (dd, 1H, J 2'',1''= 1.9 Hz, J 2'',3'' = 3.0Hz, H2''), 4.07 (dd, 0.2H, J 2β,1β = 0.9 Hz, J 2β,3β 2.9 Hz, H2β), 4.04 (dd, 1H, J 2α,1α = 2.0 Hz, J 2α,3α = 2.8 Hz, H2α), 3.90 (m, 0.8H, H5α), 3.80 (m, 1H, H5'), 3.74 (m, 1H, H5''), 3.62 (dd, 1H, J 4α,5α = J 4α,3α = 9.2 Hz, H4'), 3.58 (dd, 0.8H, J 4α,5α = J 4α,3α = 9.3 Hz, H4α), 3.56 - 3.49 (m, 1H, H3α and H4β), 3.45 (m, 1H, H3'), 3.49 - 3.48 (m, 4H, OCH3'', H4''), 3.46 (s, 3H, OCH3'), 3.45 (s, 3H, OCH3), 3.39 (m, 0.2H, H5β), 3.33 - 3.29 (dd, 1.2H, J 3'',2'' = 3.0Hz, J 3'',4'' = 9.5 Hz, H3'' and H3β), 1.35 (d, 0.6H, J 6β,5β = 6.0 Hz, H6β), 1.32 (d, 3H, J 6',5' = 6.4 Hz, H6'), 1.29 (d, 2.4H, J 6α,5α = 6.3 Hz, H6α), 1.28 (d, 3H, J 6'',5'' = 6.2 Hz, H6''). 13C NMR (150 MHz, CD3OD): δ 103.2 (C1''), 103.2 (C1'), 95.5 (C1α), 95.4(C1β), 85.4 (C3β), 83.1 (C3'),82.9 (C3α), 82.0 (C3''), 80.0 (C4α), 79.4 (C4β), 79.3 (C4'), 72.6 (C4''), 71.9(C5β), 70.5 (C5''), 69.0 (C5'), 68.6 (C2β), 68.5 (C2α), 68.3 (C2''), 68.0 (C2'), 67.7(C5α), 57.3 (OCH3''), 56.7 (OCH3α), 57.6 (OCH3'), 56.5 (OCH3β), 18.8 (C6' and C6β),18.6 (C6α), 17.9 (C6''). J C1', H1'α = J C1'', H1'' = 173 Hz, J C1α, H1α = 172 Hz, J C1 β, H1β = 163 Hz J C1', H1'α = 170 Hz LRMS m / z C 21 H 38 NaO 13 [M+Na] + Calculated, 521.2; Found, 521.2. The NMR spectrum is shown in FIG.

[0206] 3-O-methyl-α-D-rhamnopyranoside-(1→4)-3-O-methyl-α-D-rhamnopyranoside-(1→4)-3-O-methyl-α-D-rhamnopyranose (tetrasaccharide) Compound 19 tetra (22.6 mg, 26.0 μmol) was dissolved in 10 mL of anhydrous MeOH and the solution was stirred under a nitrogen gas atmosphere. Sodium metal (10 mg, 0.44 mmol) was then added and the reaction was stirred after which it reached completion as determined by TLC. The solution was then eluted with Rexin-H + Neutralization with resin, filtration through Celite, and evaporation gave the tetrasaccharide compound as a clear solid (15.9 mg, 24.1 μmol, 93%). Rf = 0.45 (MeOH / CH2Cl2, 1 / 4) [α] D25 7.7 (c 0.63, MeOH) 1 H NMR (600MHz, CD3OD): δ 5.08 (m, 3H, H1', ​​H1'',H1'''), 5.00 (d, 0.8H, J 1α,2 = 1.8 Hz, H1α), 4.66 (d, 0.2H, J 1β,2 = 0.8 Hz, H1β), 4.05 (m, 2H, J 2',1' = 2.2 Hz, J 2',3' = 2.6Hz, H2' and H2''), 4.04 (dd, 1H, J 2'',1'' = 1.9Hz, J 2'',3'' = 3.0 Hz, H2'''), 4.01 (dd, 0.2H, J 2β,1β = 0.8 Hz, J 2β,3β 3.0 Hz, H2β), 3.96 (dd, 1H, J 2α,1α = 1.9 Hz, J 2α,3α = 2.8 Hz, H2α), 3.83 (m, 0.8H, H5α), 3.76-3.69 (m, 2H, H5' and H5''), 3.67 (m, 1H, H5'''), 3.56-3.53 (m, 2H, H4' and H4''), 3.51 (dd, 0.8H, J 4α,5α = J 4α,3α = 9.3 Hz, H4α), 3.52-3.49 (m, 1H, H3α and H4β), 3.46 (dd, J 3',2' = 3.0 Hz,J 3',4' = 9.3 Hz, 1H, H3'), 3.42-3.40 (m, 4H, OCH3''',H4'''), 3.40-3.35 (s, 11H, OCH3', OCH3'', OCH3,H3'' and びH3'), 3.33 (m, 0.2H, H5β), 3.25-3.22 (m, 1.2H, H3''' and びH3β), 1.28-1.21 (m, 9H, H6β, H6', H6α and びH6''), 1.20 (d, 3H, J 6''',5''' = 6.2 Hz, H6'''). 13CNMR (150 MHz, CD3OD): δ 103.2 (C1'''), 103.0(C1''), 103.0 (C1') 95.5 (C1α), 95.4 (C1β), 85.4 (C3β), 83.0 (C3'), 83.0 (C3'') 82.9(C3α), 82.0 (C3'''), 80.0 (C4α),79.7 (C4β), 79.4 (C4'), 79.2 (C4''), 72.6 (C4'''), 71.9(C5β), 70.5 (C5'''), 69.0 (C5'), 68.9 (C5''), 68.6 (C2β), 68.2 (C2α), 68.3 (C2'''), 68.0 (C2' and C2''), 67.7 (C5α), 57.3 (OCH3'''),56.7 (OCH3α), 56.7 (OCH3'), 56.6(OCH3''), 56.5 (OCH3β), 18.8 (C6α), 18.7 (C6β), 18.5 (C6' and C6''), 17.9 (C6'''). J C1', H1' = J C1'', H1'' = J C1''',H1''' = 173 Hz, J C1α, H1α = 174 Hz, J C1 β, H1β = 162 Hz, LRMS m / z C 28 H 50 NaO 17 [M+Na] + Calculated, 681.3; Found, 680.9. The NMR spectrum is shown in FIG.

[0207] 3-O-methyl-α-D-rhamnopyranoside-(1→4)-3-O-methyl-α-D-rhamnopyranoside-(1→4)-3-O-methyl-α-D-rhamnopyranoside-(1→4)-3-O-methyl-α-D-rhamnopyranose (pentasaccharide) Compound 19 pentasaccharide (33.1 mg, 30.9 μmol) was dissolved in 10.0 mL of anhydrous MeOH and the solution was stirred under a nitrogen gas atmosphere. Sodium metal (10.0 mg, 0.44 mmol) was then added and the reaction was stirred before reaching completion as determined by TLC. The solution was then neutralized with lexan, filtered through Celite, and evaporated to give the pentasaccharide compound as a clear solid (25.3 mg, 30.9 μmol, 100%). Rf = 0.45 (MeOH / CH2Cl2, 1 / 4) [α] D 25 12.6 (c 1.0, MeOH) 1 H NMR (600MHz, CD3OD): δ 5.12 (m, 4H, H1', ​​H1'',H1''', H1''''), 5.03 (d, 0.8H, J 1α,2 = 1.8 Hz, H1α), 4.71 (s, 0.2H, H1β), 4.09 (m, 3H, H2' and H2'', H2'''), 4.04 (dd,1H, J 2'''',1'''' = 1.9 Hz, J 2'''',3'''' = 2.8 Hz,H2''''), 4.05 (dd, 0.2H, J 2β,1β = 0.8 Hz, J 2β,3β 2.8 Hz, H2β), 4.00 (dd, 1H, J 2α,1α = 2.0 Hz, J 2α,3α = 2.6 Hz, H2α), 3.87 (m, 0.8H, H5α), 3.80-3.73 (m, 3H,H5', H5'' and H5'''), 3.71 (dd, 1H, J 5'''',4'''' =9.1Hz, J 5'''',6'''' = 6.3 Hz, H5''''), 3.60-3.56 (m, 3H, H4', H4'',H4'''), 3.55 (dd, 0.8H, J 4α,5α = J 4α,3α= 9.3 Hz, H4α), 3.52 - 3.47 (m, 0.8H, H3α and H4β), 3.46 - 3.39 (m, 19.2H, OCH3x5, H4'''', H3', H3'' and H3''', H5β), 3.29 (m, 1.2H, H3β and H3''''), 1.23 - 1.22(m, 12H, H6β, H6', H6α, H6'',H6'''), 1.24 (d, 3H, J 6'''',5'''' = 6.1 Hz, H6''''). 13 13C NMR (151 MHz, CD3OD): δ 103.2 (C1''''),103.1 (C1'''), 103.0 (C1'' and C1'), 95.5 (C1α), 95.3 (C1β), 85.3 (C3β), 83.0 (C3'), 83.0 (C3''), 83.0 (C3'''), 82.9 (C3α), 82.0 (C3''''), 80.0 (C4α), 79.5 (C4β and C4'), 79.4 (C4''), 79.2 (C4'''), 72.6 (C4'''') 71.9 (C5β), 70.5 (C5''''), 69.0 (C5'), 69.0 (C5''), 69.0 (C5'''), 68.6 (C2β), 68.4 (C2α), 68.3 (C2''''), 68.0 (C2',C2'' and C2'''), 67.7 (C5α), 57.3(OCH3''''), 56.7 (OCH3α, OCH3',OCH3'', OCH3'''), 56.6 (OCH3β), 18.8 (C6α), 18.6 (C6β, C6', C6'' and C6'''), 17.9 (C6''''). J C1',H1' = J C1'', H1'' = J C1''', H1''' = J C1'''',H1'''' = 173 Hz, J C1α, H1α = 170 Hz, J C1 β, H1β = 160 Hz, LRMS m / z C 35 H 62 NaO 21 [M + Na] + calculated value of, 841.4; measured value, 841.3. The NMR spectrum is shown in Figure 20.

[0208] 2-[N-(tert-butoxycarbonyl)amino]ethyl 2-O-acetyl-4-O-benzyl-3-O-methyl-α-D-rhamnopyranoside (21) Compound 16 (46.4 mg, 111 μmol) and N-boc-ethanolamine (19 μL, 123 μmol) were coevaporated with toluene (5×10 mL) and dried under high vacuum overnight. Anhydrous CHCl (5 mL) was added, followed by 0.1 g of activated powdered 3 Å molecular sieves. The reaction was then cooled to −78° C. under a nitrogen atmosphere and N-iodosuccinimide (33.7 mg, 150 μmol) was added, followed by triflic acid (10 μL, 115 μmol). The reaction was stirred for 3 hours until completion was reached. The reaction was then filtered through a Buchner funnel, diluted with 10.0 mL of methylene chloride, and washed with 10% NaSO (2×10 mL) and saturated sodium bicarbonate solution (1×10 mL). The organic layer was then dried over Na2SO4, filtered and purified by flash chromatography (eluent: EtOAc / Hexanes) to give compound 21 as a clear oil (42.0 mg, 88 μmol, 80%). Rf = 0.30 (EtOAc / Hexanes, 3 / 7) [α] D 25 108.8 (c 22.5, CHCl3) 1 HNMR α, (500 MHz, CDCl3): δ 7.37-7.32 (m, 4H, Bn), 7.31-7.28 (m, 1H, Bn), 5.29 (dd, 1H, J 2,1 = 1.3 Hz, J 2,3 = 3.1 Hz, H2), 4.88 (d, 1H, J A,B = 10.9Hz, CHA, OBn), 4.85 (br. s, 1H, NH), 4.71 (d, 1H, J 1,2 = 1.3 Hz,H1), 4.60 (d, 1H, J B,A = 10.9 Hz, CHB, OBn), 3.73-3.66 (m, 2H, H5 and CHA, CH2CH2NHBoc), 3.63 (dd, 1H, J 3,2 = 3.4 Hz, J3,4 = 9.4 Hz, H3), 3.50-3.44 (m, 1H, CHB, CH2CH2NHBoc),3.44 (s, 3H, CH3O), 3.39-3.32 (m, 2H, H4 and CHA,CH2CH2NHBoc), 3.30-3.22 (m, 1H, CHB, CH2CH2NHBoc),2.15 (s, 3H, Ac), 1.45 (s, 9H, C(CH3)3, Boc), 1.32 (d,3H, J 6,5 = 6.3 Hz, H6). 13 C NMR (125 MHz, CDCl3):δ = 170.5, 155.9 (C=O), 138.6, 128.5 (x2), 128.0 (x2),127.8 (Bn), 97.8 (C1), 80.1 (C4), 79.9 (C3), 79.5 (CC(CH3)3),75.4 (CH2, Bn), 68.6 (C2), 67.8 (C5), 67.3 (CH2CH2NHBoc),57.6 (OCH3), 40.3 (CH2CH2NHBoc), 28.5 ((CH3)3,BOC), 21.1 (Ac), 18.0 (C6). HRMS m / z C 23 H 35 NNaO8[M+Na] + Calculated value: 476.2255; measured value: 476.2249.

[0209] 2-[N-(tert-butoxycarbonyl)amino]ethyl 2-O-acetyl-4-O-benzyl-3-O-methyl-α-D-rhamnopyranoside (22) Compound 21 (46 mg, 101.5 μmol) was dissolved in MeOH (5 mL) and charged with palladium on carbon (40 mg, 0.38 mmol). The solution was then bubbled with a balloon of hydrogen gas for 5 min and stirred continuously at room temperature under hydrogen gas atmosphere. After 4 h, the reaction reached completion and was filtered through Celite and evaporated to give pure 22 as a clear oil (21.0 mg, 57.8 μmol, 62%). Rf = 0.35 (EtOAc / Hexane, 1 / 1), 1HNMR (500 MHz, CDCl3): δ 5.30 (dd, 1H, J 2,1 = 1.8 Hz, J 2,3 = 3.1 Hz, H2), 4.78 (br. s, 1H, NH), 4.74 (d,1H, J 1,2 = 1.6 Hz, H1), 3.76-3.65 (m, 2H, H5 and CHA, CH2CH2NHBoc), 3.53-3.43 (m, 3H, H3, H4 and CHB, CH2CH2NHBoc), 3.41 (s, 3H, CH3O),3.39-3.26 (m, 2H, CH2CH2NHBoc), 2.34 (br. s, OH), 2.12(s, 3H, Ac), 1.46 (s, 9H, C(CH3)3, Boc), 1.34 (d, 3H, J 6,5 = 6.2 Hz, H6). 13 C NMR (125 MHz, CDCl3): δ = 170.3, 155.9 (C=O), 98.0 (C1), 79.4 (C4), 71.6 (C3), 68.3 (C2),67.5 (C5), 67.2 (CH2CH2NHBoc), 57.3 (OCH3),40.2 (CH2CH2NHBoc), 28.4 ((CH3)3,BOC), 20.9 (Ac), 17.7 (C6). HRMS m / z C 16 H 29 NNaO8[M+Na] + Calculated value: 386.1791; measured value: 386.1784.

[0210] 2-[N-(tert-butoxycarbonyl)amino]ethyl 2-O-acetyl-4-O-benzyl-3-O-methyl-α-D-rhamnopyranoside-(1→4)-2-O-acetyl-3-O-methyl-α-D-rhamnopyranoside (23) Compound 22 (21.0 mg, 57.8 μmol) and compound 16 (28.9 mg, 69.4 μmol) were coevaporated with toluene (5×10 mL) and dried under high vacuum overnight. Anhydrous CHCl (5 mL) was added followed by 0.1 g of activated powdered 3 Å molecular sieves. The reaction was then cooled to −78° C. under a nitrogen atmosphere and N-iodosuccinimide (19.1 mg, 84.9 μmol) was added followed by triflic acid (5.0 μL, 57.5 μmol). The reaction was stirred for 3 hours until completion was reached. The reaction was then filtered through a Buchner funnel, diluted with CHCl (10 mL) and washed with 10% NaSO (2×10 mL) and saturated NaHCO solution (1×10 mL). The organic layer was then dried over Na2SO4, filtered and purified by flash chromatography (eluent: EtOAc / Hexanes) to give compound 23 as a clear oil (18 mg, 26.5 μmol, 47%). (Rf = 0.65 EtOAc / Hexanes 3 / 7). 1 HNMR α, (500 MHz, CDCl3): δ 7.38-7.32 (m, 4H, Bn), 7.31-7.27 (m, 1H, Bn), 5.35 (m, 1H, H2'),5.28 (m, 1H, H2), 5.12 (s, 1H, H1'), 4.88 (d, 1H, J A,B = 10.8 Hz,CHA, OBn), 4.83 (br. s, 1H, OH), 4.69 (s, 1H, H1), 4.62 (d, 1H, J B,A= 10.9 Hz, CHB, OBn), 3.85-3.77 (m, 1H, H5'), 3.75-3.68 (m, 1H, CHA, CH2CH2NHBoc),3.66-3.61 (m, 1H, H5), 3.61-3.56 (m, 1H, H3'), 3.56-3.51 (m, 4H, H3 and H4), 3.51-3.42 (m, 4H, CHB, CH2CH2NHBoc and CH3O), 3.41-3.34 (m, 4H, H4' and CH3O),3.33-3.24 (m, 2H, CH2CH2NHBoc), 3.30 (bs, 1H, NH) 2.13,2.10 (2 xs, 6H, Ac x 2), 1.46 (s, 9H, C(CH3)3, Boc), 1.33-1.27 (m, 6H, H6 and H6'). 13 C NMR (125MHz, CDCl3): δ = 170.4, 170.2 155.9 (C=O),138.6, 128.4 (x2), 128.0 (x2), 127.8 (Bn), 99.4 (C1'), 97.8 (C1), 80.0 (C4'),80.0 (C4), 79.9 (C3'), 79.6 (C(CH3)3), 78.3 (C3), 75.4(CH2, Bn), 68.7 (C2'), (C5'), 67.9 (C2), 67.3 (CH2CH2NHBoc),67.1 (C5), 57.6 and 57.4 (OCH3), 40.3 (CH2CH2NHBoc),28.5 ((CH3)3, BOC), 21.2 and 21.1 (Ac), 18.2 and 17.9 (C6 and C6'). C1',H1'α = 171 Hz, J C1, H1α = 168 Hz HRMS: m / z C 32 H 49 NNaO 13 [M+Na] + Calculated for, 678.3102; Found, 678.3092. The NMR spectrum is shown in FIG.

[0211] 2-Aminoethyl-3-O-methyl-α-D-rhamnopyranoside-(1→4)-3-O-methyl-α-D-rhamnopyranose (linker disaccharide) Compound 23 (18 mg, 27 μmol) was dissolved in MeOH (2 mL), PD / C (10 mg, 93 μmol) was added, and the mixture was stirred under a hydrogen atmosphere by balloon pressure for 16 h. The compound was then diluted with MeOH (10 mL), filtered through Celite, and concentrated. The concentrate was then dissolved in MeOH (2 mL) and solid sodium (10 mg, 435 μmol) was added. The reaction was stirred under a nitrogen atmosphere for 16 h and lexin-H + Resin was added until a pH of 5 was reached. The mixture was filtered and the filtrate was concentrated. The compound was then dissolved in DCM (2 mL) and TFA (160 μL) was added at 0° C. After 30 min at 0° C., the DCM and TFA mixture was evaporated to give the linker disaccharide as a yellow oil (10.3 mg, 27 μmol, 100% yield). (Rf = 0.12, DCM / MeOH / water / NH4OH, 60 / 35 / 6 / 1). 1 H NMR (600 MHz, D2O): δ 5.16 (d, 1H, J 1,2 = 1.6 Hz, H1'), 4.87 (d, 1H, J 1,2 = 1.7 Hz, H1), 4.23 (m, 2H, H2' and H2) 3.97 (m, 1H,CHACH2NH2), 3.80 (m, 2H, H5' and H5), 3.70 (m, 1H, CHBCH2NH2), 3.64 (dd, 1H, J 3,2 =3.3 Hz, J 3,4 = 9.2 Hz, H3), 3.58 (dd, 1H, J 4,3 =J 4,5 = 9.2 Hz, H4), 3.50 (dd, 1H, J 4',3' = J 4',5' = 9.6 Hz, H4'), 3.44 (s, 3H, OCH3), 3.44 (s, 3H, OCH3),3.41 (dd, 1H, J 3',2' = 3.3 Hz, J 3',4' = 9.7Hz, H3'), 3.27 (m, 2H, CH2NH2), 1.33 (d, 3H, J 6,5 = 5.9 Hz, H6), 1.29 (d, 3H, J 6',5'= 6.4 Hz, H6'). 13 CNMR (150 MHz, D2O): δ 101.4 (C1'), 99.7(C1), 80.5 (C3), 79.4 (C3'), 78.1 (C4), 70.6 (C4'), 69.5 (C5'), 67.2 (C5), 66.0(C2'), 65.8 (C2), 63.4 (CH2CH2NH2), 56.1 (2 xOCH3), 39.0 (CH2NH2), 17.3 (C6), 16.5 (C6'). J C1',H1'α = 174 Hz, J C1, H1α = 173 Hz ESI-MS: m / z C 16 H 32 NO9[M+H] + Calculated value: 382.2072; measured value: 382.2071.

[0212] Example 3A: Results and Discussion Synthesis of 3-O-methyl D-rhamnose: D-rhamnose was first synthesized by a slight modification of the method described by Zunk et. al. and others [28-31] (Scheme 1, shown in Figure 22). Acetylation of D-mannose followed by anomeric benzylation produced the 1-O-benzyl derivative 1 in 49% yield in two steps. After crude purification of 1 and treatment with sodium methoxide, iodination at C6 gave the iodide 2 in excellent yield. Reduction of 2 with palladium hydroxide and hydrogen gas produced the D-rhamnose derivative 3 in 100% yield. 1The doublet at 1.28 ppm in the H NMR spectrum confirmed the dehydroxylation at C6. 2,3-O-acetonide protection of the D-rhamnose derivative 3, followed by O-4 benzylation, gave 4 in 90% yield in two steps. Acetonide deprotection gave the diol 5. Selective methylation of 3-O by treatment with dibutyltin oxide and reaction with cesium fluoride and methyl iodide gave 6 in excellent yield (91%) in two steps

[32] . Long-range HMBC coupling between the O-methyl singlet and C3 confirmed the regioselective introduction of the methyl. Global deprotection of intermediate 6 gave 3-O-methyl D-rhamnose 7 in 59% yield in two steps. (HRMS m / z C7H 14 NaO5[M+Na] + Calculated value, 201.0739; measured value, 201.0734).

[0213] Synthesis of 3-O-methyl L-rhamnose: Naturally abundant and inexpensive L-rhamnose was converted to the thioglycoside donor 8 in excellent yield after acetylation and reaction with thiocresol [31-36]. Deacetylation and acetonide protection under Zemplen conditions gave intermediate 9. Benzylation and acetonide deprotection afforded the diol 10 in excellent yield. Selective methylation of 3-O using dibutyltin oxide, followed by treatment with cesium fluoride and methyl iodide, afforded the methylated derivative 11 in 55% yield in two steps. The reaction was regioselectively confirmed by a strong long-range signal between the O-methyl singlet and C3, as previously described for intermediate 6. N-bromosuccinimide in acetone and water afforded the hemiacetal 12 in 71% yield. Acetylation, followed by benzyl reduction and subsequent deacetylation afforded 3-O-methyl L-rhamnose 13 in 24% yield in three steps (HRMS m / z C7H 14 NaO5[M+Na] + Calculated value, 201.0733; Measured value, 201.0735) (Scheme 2, shown in Figure 23).

[0214] Synthesis of 3-O-methyl D-rhamnose oligosaccharides: Anomeric deprotection of 6 with HCl, acetonitrile mixture using the resulting D-enantiomer-pure intermediate 6 produced hemiacetal 14 in 65% yield. 14 was then acetylated and treated with thiocresol and BF3OEt2 to produce donor 16 in good yield. Then, after hydrogenation of benzylated intermediate 15, acceptor 17 was produced in excellent yield (96%). Subsequent glycosylation reaction between 16 and 17 activated with N-iodosuccinimide and triflic acid produced disaccharide 18di in 53% yield (α only). In addition, the coupling was confirmed by the long-range HMBC signal between H1' and C4. The resulting additional adjacent C1',H1' coupling constant (J C1’,H1’ = 179 Hz), indicating alpha configuration, consistent with previous literature data

[34] . 1 After benzyl deprotection, the disaccharide acceptor 19di was formed, as indicated by the absence of signals in the benzyl region of the H NMR spectrum. After repeated rounds of iterative glycosylation and hydrogenation, the pentasaccharide 18penta was finally obtained. Each iterative glycosylation afforded only minor amounts of the respective oligosaccharide, as shown in Scheme 3 (shown in Figure 24). Despite the length of the oligosaccharide, benzyl removal maintained consistently high yields (90-100%). Final deprotection of 19penta under Zemplen conditions afforded the target pentasaccharide 1 in 100% yield, with an overall yield of 1.0% from D-mannose. Target 19di, target 19tri, and target 19tetra were also deprotected under Zemplen conditions to afford compound 20di, compound 20tri, and compound 20tetra in excellent yields.

[0215] 2-Aminoethyl Handle Glycosylation: A common approach for conjugating oligosaccharides to proteins to generate conjugate vaccines is to glycosylate the reducing end of the handle-bearing oligosaccharide [37,38]. Glycosylation of intermediate 16 with N-boc ethanolamine gave intermediate 21 in good yield (shown in Scheme 4, Figure 25). In addition, the long-range HMBC signal between H1 and the adjacent linker CH2 confirmed the coupling. The resulting adjacent C1,H1 coupling constants were consistent with an α-configuration

[34] . Benzyl deprotection with Pd / C gave compound 22 in 62% yield, and subsequent glycosylation of compound 22 with 16 produced 23 in 53% yield. Global deprotection of 23 produced the final target 24 in 100% yield.

[0216] Example 4: Pseudomonas aeruginosa methylated rhamnan synthetic pentasaccharide conjugation with HSA (without handle or linker) 1.25 mg of HSA in water was added to methylated rhamnan synthetic pentasaccharide (without handle or linker) produced according to Example 3 in 300 μl of 20% methanol. It was then left at room temperature for 1 hour and freeze-dried. The freeze-dried material was immediately dissolved in 300 μl of 0.2 M sodium borate containing 0.5 M sodium sulfate and 15 mg / ml sodium cyanoborohydride and left at 55° C. for 72 hours. The sample was converted to water using a Millipore Ultra-15 30K MWCO spin column (3× spin with water). An aliquot was checked by MALDI and SDS-PAGE and the remainder was freeze-dried (1.1 mg) and stored at −20° C. until used for immunization of mice. Six Balb / C mice were immunized with a prime-one-boost-two schedule of glycoconjugates and adjuvants. Sera after the second boost were tested for cross-reactivity to LPS and whole cells from P. aeruginosa by ELISA.

[0217] On SDS-PAGE, conjugation was illustrated by a change in migration of the HSA molecule versus the conjugate (Figures 26A and 26B). MALDI analysis indicated that approximately six pentasaccharides were conjugated to HSA, with a mass increase of approximately 5 kDa when each pentasaccharide unit is 872 amu (Figure 27). ELISA analysis showed that the resulting sera recognized LPS from P. aeruginosa (Figures 28A-28F) and killed whole cells (Figure 29), highlighting the ability of the synthetic pentasaccharide glycoconjugate to achieve the response required to recognize the target P. aeruginosa epitope, as detailed for whole cells.

[0218] Example 5: Synthesis of 3-O-methyl tri-, tetra-, and penta-rhamnose oligosaccharides with handles and optional linkers See Figure 30. The Boc-protected aminoethyl handle was coupled to the thiotoluene intermediate S8 (same as 16 above) in 84% yield. Subsequent removal of the benzyl protecting group under hydrogenation conditions afforded donor S10 (same as 22 above) in excellent yield. Repetitive 1,4 glycosylation with thiotoluene to protect S8 (same as 16 above) followed by 4-O-benzyl deprotection produced S11 (same as 23 above), S13, S15, and S17. Quantitative 4-O-benzyl deprotection of S13, S15, and S17 was followed by deacetylation using sodium methoxide and Boc deprotection with TFA to afford S19 (same as 24 above), S20, and S21. Without further purification, such compounds were conjugated with bivalent linkers containing activated N-hydroxysuccinimide and aldehyde-protected hemiacetals after semi-preparative reverse-phase purification (see Scheme 5, Figure 30).

[0219] [ka] p-Tolyl 1-thio-α-D-mannopyranoside (S1) In a 2000 mL round bottom flask, acetylated mannose (142.5 g, 365.1 mmol) was dissolved in 800 mL of anhydrous CHCl and the solution was cooled to 0° C. under N. Thiocresol (63.5 g, 511.0 mmol) was then added, followed by boron trifluoride diethyl etherate (63.1 mL, 511.0 mmol) and the solution was stirred at RT for 48 h until TLC indicated completion. The reaction was then washed with water (2×200 mL) and NaHCO (2×200 mL). The organic layer was isolated, dried over NaSO, filtered and evaporated under reduced pressure. The crude product was then dissolved in 1000 mL of anhydrous MeOH and cooled to 0° C. Sodium metal (0.873 g, 36.4 mmol) was then added and the reaction was stirred at RT for 30 h until completion was reached. The solution was diluted with DowexH + The mixture was neutralized with hexanes, filtered, and evaporated. The crude product was then dissolved in water (500 mL) and extracted with CH2Cl2 (3 x 150 mL). The aqueous layer was then evaporated to give S1 as a pale green powder (96.86 g, 245 mmol, 67%, over two steps). Rf = 0.65 (MeOH / CH2Cl2, 15 / 85, V / V)[α] D 25 2.5 (c 0.16, MeOH) 1 HNMR (400 MHz, CD3OD): δ 7.39 (d, 2H), 7.11(d, 2H), 5.33 (s, 1H), 4.08-3.98 (m, 2H), 3.83-3.62 (m, 3H), 3.29 (s, 1H), 2.29(s, 3H) 13 C NMR (100 MHz, CD3OD): δ 138.9, 133.5 (2C), 132.1, 130.8 (2C), 90.8, 75.5, 73.7, 73.1, 68.7,62.6, 21.1 ESI-MS: m / z C 13 H 18 O5NaS [M+Na] + Calculated value, 309.0767; measured value, 309.0769.

[0220] [ka] p-Tolyl 6-iodo-1-thio-α-D-mannopyranoside (S2) Compound S1 (96.86 g, 338.2 mmol) was dissolved in anhydrous THF (600 mL) and the solution was refluxed under N2 at 60 °C. Triphenylphosphine (133.05 g, 507.3 mmol), imidazole (46.04 g, 676.4 mmol), and I2 (128.76 g, 507.3 mmol) were then slowly added. The reaction was complete after 10 min when the purple color of iodine persisted. The solution was then cooled to RT and concentrated under reduced pressure. The crude mixture was dissolved in ethyl acetate (600 mL) and washed with 10% Na2S2O3 (2 x 150 mL) and water (2 x 150 mL). The organic layer was then dried over Na2SO4, filtered, evaporated and purified by flash chromatography (eluent: MeOH / CH2Cl2, 1 / 5, V / V) to give compound S2 as a white solid (87.37 g, 220.5 mmol, 65%). Rf = 0.65 (MeOH / CH2Cl2, 1 / 9, V / V) [α] D 25 -2.1 (c 0.041, MeOH) 1 H NMR(400 MHz, CD3OD): δ 7.47 (d, 2H), 7.14 (d,2H), 5.34 (s, 1H), 3.93 (dd, 1H), 3.67-3.52 (m, 3H), 3.31 (s, 1H), 2.32 (s, 3H) 13 C NMR (100 MHz, CD3OD): δ138.9, 133.4 (2C), 132.2, 130.7 (2C), 91.0, 74.9, 73.8, 72.7, 72.6, 21.1, 6.0ESI-MS: m / z C 13 H 17 O4INaS [M+Na] + Calculated value: 418.9784; Measured value: 418.9786.

[0221] [ka] p-Tolyl 1-thio-α-D-rhamnopyranoside (S3) Compound S2 (1.76 g, 4.44 mmol) was dissolved in MeOH (30 mL), the solution was charged with Pd(OH)2 (0.39 g, 2.76 mmol) and the solution was aerated with H2. N,N-diisopropylethylamine (1.8 mL, 10.3 mmol) was then added and the solution was stirred under H2 atmosphere for 6 h until completion was reached. The solution was then filtered through a pad of Celite, evaporated and purified by flash chromatography (eluent: MeOH / CHCl2, 1 / 5, V / V) to give compound S3 as a clear oil (0.65 g, 2.40 mmol, 54%). Rf = 0.50 (MeOH / CHCl2, 1 / 9, V / V) [α] D 25 0.91 (c 0.043, MeOH) 1 H NMR(400 MHz, CD3OD): δ 7.35 (d, 2H), 7.14 (d,2H), 5.29 (s, 1H), 4.10-4.00 (m, 2H), 3.64 (dd, 1H), 3.45 (dd, 1H), 3.31 (s,1H), 2.31 (s, 3H), 1.26 (d, 3H) 13 C NMR (100 MHz, CD3OD):δ 138.8, 133.3 (2C), 132.2, 130.8 (2C), 90.6, 74.2,73.8, 72.9, 72.6, 21.1, 17.8 ESI-MS: m / z C 13 H 18 O4NaS[M+Na] + Calculated value, 293.0818; Measured value, 293.0818.

[0222] [ka] p-Tolyl 2,3-O-isopropylidene-1-thio-α-D-rhamnopyranoside (S4) Compound S3 (10.16 g, 37.6 mmol) was dissolved in 250 mL of anhydrous acetone. To this solution, 2,2-dimethoxypropane (8.29 mL, 67.6 mmol) and p-toluenesulfonic acid monohydrate (0.71 g, 0.376 mmol) were added and the reaction was stirred at RT for 3 h until it reached completion. The solution was evaporated under reduced pressure, dissolved in ethyl acetate (300 mL) and washed with saturated NaHCO3 (2 x 100 mL). The combined organic layers were dried over Na2SO4, filtered and purified by flash chromatography (eluent: EtOAc / hexane, V / V) to give compound S4 as a white solid (10.68 g, 34.4 mmol, 92%). Spectral data was consistent with literature values. 1

[0223] [ka] p-Tolyl 4-O-benzyl-2,3-O-isopropylidene-1-thio-α-D-rhamnopyranoside (S5) Compound S4 (10.68 g, 34.4 mmol) was dissolved in anhydrous DMF (150 mL) and the solution was cooled to 0° C. NaH (2.06 g, 51, 6 mmol) was then added in small portions and the solution was stirred for 30 min. Benzyl bromide (4.91 mL, 41.3 mmol) was added dropwise and the solution was stirred at RT for 2 h until completion was reached. The reaction was quenched by the addition of Et3N (10 mL) and then poured into a cooled solution of saturated NH4Cl (400 mL). The solution was stirred at 0° C. for 1 h and then filtered. The solid was collected and dried under high vacuum to give S5 as a beige solid (13.43 g, 33.5 mmol, 98%). Spectroscopic data was consistent with literature values. 2

[0224] [ka] p-Tolyl 4-O-benzyl-1-thio-α-D-rhamnopyranoside (S6) Compound S5 (1.09 g, 2.72 mmol) was dissolved in an 80% (v / v) solution of AcOH (10 mL) and stirred overnight at RT. The solution was then heated to 55° C. in an oil bath for 3 h to drive the reaction to completion. The solution was then evaporated under reduced pressure and co-evaporated with toluene (4×30 mL) to give S6 as a clear oil (909 mg, 2.52 mmol, 92%). Spectral data was consistent with literature values. 2

[0225] [ka] p-Tolyl 4-O-benzyl-3-O-methyl-1-thio-α-D-rhamnopyranoside (S7) Compound S6 (14.3 g, 39.7 mmol) was coevaporated with toluene (3×100 mL) and dried under high vacuum overnight. S6 was then dissolved in anhydrous toluene (400 mL), purged with N2 gas, and charged with dibutyltin(IV) oxide (11.85 g, 47.6 mmol). The reaction mixture was then stirred at reflux for 16 h, cooled to RT, and evaporated under reduced pressure. The crude tin acetal was left under high vacuum for 5 h, dissolved in anhydrous DMF (793 mL), and purged with N2. Cesium fluoride (9.00 g, 59.6 mmol) and iodomethane (28.15 mL, 396.6 mmol) were added, and the reaction was stirred at 40° C. for 16 h under N2. The mixture was cooled to RT, diluted with EtOAc (350 mL), and washed with water (5×150 mL), saturated NaHCO3 (150 mL), and brine (150 mL). The combined organic layers were then dried over Na2SO4, evaporated, and purified by flash chromatography (eluent: EtOAc / Hexane, V / V) to give S7 as a clear oil (10.87 g, 29.0 mmol, 73%). Rf = 0.40 (EtOAc / Hexane, 3 / 7, V / V) [α] D 25 8.2 (c 0.38, CHCl3) 1HNMR (400 MHz, CD3Cl): δ 7.40-7.27 (m, 8H),7.11 (d, 2H), 5.47 (d, 1H), 4.86 (d, 1H). 4.64 (d, 1H), 4.30 (dd, 1H), 4.20(dq, 1H), 3.58 (dd, 1H), 3.52 (s, 3H), 3.44 (dd, 1H), 2.32 (s, 3H), 2.28 (br.s, 1H), 1.30 (d, 3H) 13 C NMR (100 MHz, CD3Cl): δ 138.6. 137.8, 132.2 (2C), 130.4, 130.0 (2C), 128.6 (2C), 128.1(2C), 127.9, 87.6, 82.1, 80.2, 75.4, 69.5, 68.6, 57.6, 21.2, 17.9 ESI-MS: m / z C 21 H 26 O4NaS[M+Na] + Calculated value, 397.1444; measured value, 397.1444.

[0226] [ka] p-Tolyl 2-O-acetyl-4-O-benzyl-3-O-methyl-1-thio-α-D-rhamnopyranoside (S8 (same as 16 above)) Compound S7 (10.85 g, 29.0 mmol) was dissolved in acetic anhydride (20 mL) and pyridine (20 mL) and the solution was stirred at RT for 16 h. The solution was then evaporated under reduced pressure and coevaporated with toluene (5×30 mL). The crude oil was then purified by flash chromatography (eluent: EtOAc / hexane, V / V) to give compound S8 as a clear oil (10.77 g, 25.9 mmol, 89%). Spectral data was consistent with literature values. 3

[0227] [ka] 2-(tert-Butyloxycarbonylamido)ethyl 2-O-acetyl-4-O-benzyl-3-O-methyl-α-D-rhamnopyranoside (S9 (same as 21 above)) Compound S8 (46.4 mg, 0.11 mmol) and N-boc-ethanolamine were coevaporated with toluene (5×10 mL) and dried under high vacuum overnight. Anhydrous CHCl (5 mL) was added, followed by 0.1 g of activated powder 3 Å molecular sieves, and the reaction was cooled to −78° C. under N gas atmosphere. N-iodosuccinimide (33.7 mg, 0.12 mmol) was then added, followed by triflic acid (10.0 μL, 0.12 mmol), and the reaction was stirred for 3 hours until completion was reached. The mixture was filtered through a Buchner funnel, diluted with CHCl (10 mL), and washed with 10% NaSO (2×10 mL) and saturated NaHCO (10 mL). The organic layer was then dried over Na2SO4, filtered, and purified by flash chromatography (eluent: EtOAc / Hexane, V / V) to give compound S9 as a clear oil (42.0 mg, 0.093 mmol, 84%). Rf = 0.55 (EtOAc / Hexane, 1 / 1, V / V) [α] D 25 108.8 (c 2.3, CHCl3) 1 HNMR (500 MHz, CDCl3): δ 7.45-7.36 (m, 5H),4.89 (d, 1H), 4.84 (br. s, 1H), 4.70 (s, 1H), 4.61 (d, 1H), 3.72-3.65 (m, 2H),3.63 (dd, 1H), 3.49-3.44 (m, 1H), 3.42 (s, 3H), 3.36-3.30 (m, 1H), 3.35 (dd,1H), 3.30-3.21 (m, 1H), 2.14 (s, 3H), 1.44 (s, 9H), 1.31 (d, 3H) 13CNMR (150 MHz, CDCl3): δ 170.5, 155.9, 138.6,128.5 (2C), 128.0 (2C), 127.8, 97.8, 80.1, 79.9, 75.4, 68.6, 67.8, 67.3, 57.6,40.3, 28.5, 21.1, 18.0 ESI-MS: m / z C 23 H 35 NO8H[M+H] + Calculated value, 454.2435; measured value, 454.2435.

[0228] [ka] 2-(tert-Butyloxycarbonylamido)ethyl 2-O-acetyl-3-O-methyl-α-D-rhamnopyranoside (S10 (same as 22 above)) Palladium hydroxide on carbon (1.00 g, 7.00 mmol) was added to MeOH (20 mL) and the solution was bubbled with H2 for 30 min. Compound S9 (1.37 g, 3.02 mmol) was then added and the solution was stirred at RT with a continuous flow of H2 for 3 h. The reaction was then filtered through a pad of Celite and evaporated to give S10 as a clear oil (1.07 g, 2.94 mmol, 98%). Rf = 0.35 (EtOAc / Hexane, 1 / 1, V / V) [α] D 25 9.6 (c 0.28, CHCl3) 1 HNMR (500 MHz, CDCl3 / CD3OD): δ5.29 (s, 1H), 4.78 (br. s, 1H), 4.74 (s, 1H), 3.76-3.65 (m, 2H), 3.54-3.47 (m,2H), 3.46 (dd, 1H), 3.41 (s, 3H), 3.38-3.36 (m, 2H), 2.34 (br. s, 1H), 2.11 (s,3H), 1.46 (s, 9H), 1.33 (d, 3H) 1H NMR (125 MHz, CDCl3 / CD3OD): δ 170.4, 155.9, 98.0, 79.5, 71.6,68.3, 67.5, 67.2, 57.3, 40.3, 28.4, 20.9, 17.7 ESI-MS: m / z C 16 H 29 NO8H[M+H] + Calculated value: 364.1966; measured value: 364.1963.

[0229] [ka] 2-O-acetyl-4-O-benzyl-3-O-methyl-α-D-rhamnopyranoside-(1→4)-2-(tert-butoxycarbonyl)amino]ethyl 2-O-acetyl-3-O-methyl-α-D-rhamnopyranoside (S11 (same as 23 above)) Compound S10 (1.07 g, 2.93 mmol) and compound S8 (1.47 g, 3.53 mmol) were coevaporated with toluene (5×20 mL) and dried under high vacuum overnight. Anhydrous methylene chloride (50 mL) was added followed by 0.500 g of activated powdered 3 Å molecular sieves. The reaction was then cooled to −78° C. under N2 atmosphere and N-iodosuccinimide (1.13 g, 4.11 mmol) was added followed by triflic acid (0.50 mL, 5.66 mmol). The reaction was stirred for 3 hours until completion was reached. The reaction was then filtered through a Büchner funnel, diluted with CHCl (100 mL) and washed with 10% NaSO (2×100 mL) and saturated NaHCO (100 mL). The organic layer was then dried over Na2SO4, filtered, and purified by flash chromatography (eluent: EtOAc / Hexane, V / V) to give compound S11 as a clear oil (1.56 g, 2.38 mmol, 81%). Rf = 0.65 (EtOAc / Hexane, 1 / 1, V / V) [α] D 25 66.8 (c 1.0, CHCl3) 1HNMR (400 MHz, CDCl3): δ 7.38-7.27 (m, 5H),5.35 (dd, 1H), 5.28 (s, 1H), 5.12 (d, 1H), 4.89 (d, 1H), 4.82 (br. s, 1H), 4.69(d, 1H), 4.61 (d, 1H), 3.86-3.77 (m, 1H), 3.75-3.66 (m, 1H), 3.66-3.55 (m, 1H),3.55-3.45 (m, 3H), 3.44 (s, 3H), 3.39 (s, 3H), 3.39-3.33 (m, 2H), 3.33-3.23 (m,1H), 2.13 (s, 3H), 2.10 (s, 3H), 1.46 (s, 9H), 1.34-1.28 (m, 6H) 13 CNMR (125 MHz, CDCl3): δ 170.4, 170.2, 155.9,138.6, 128.4 (2C), 128.0 (2C), 127.8, 99.4, 97.8, 80.1, 80.0, 79.9, 78.3, 75.4,68.7, 68.5, 67.9, 67.3, 67.1, 57.6, 57.4, 40.3, 28.5, 21.2, 21.0, 18.2, 17.9ESI-MS: m / z C 32 H 49 NNaO 13 [M+Na] + Calculated value, 678.3096; measured value, 678.3092.

[0230] [ka] 2-O-acetyl-3-O-methyl-α-D-rhamnopyranoside-(1→4)-2-(tert-butoxycarbonyl)amino]ethyl 2-O-acetyl-3-O-methyl-α-D-rhamnopyranoside (S12) Palladium hydroxide on carbon (1.20 g, 8.55 mmol) was added to MeOH (50 mL) and the solution was bubbled with H2 for 30 min. Compound S11 (1.71 g, 2.61 mmol) was then added and the solution was stirred at RT with a continuous flow of H2 for 3 h. The reaction was then filtered through a pad of Celite and evaporated to give S12 as a clear oil (1.47 g, 2.59 mmol, 99%). Rf = 0.20 (EtOAc / Hexane, 1 / 1, V / V) [α] D 25 1.07 (c 0.33, CHCl3) 1 HNMR (400 MHz, CDCl3): δ 5.36 (dd, 1H),5.31-5.26 (m, 1H), 5.14 (s, 1H), 4.82 (br. s, 1H), 4.70 (s, 1H), 3.84-3.75 (m,1H), 3.75-3.62 (m, 1H), 3.58-3.44 (m, 4H), 3.43-3.34 (m, 8H), 3.34-3.21 (m,1H), 2.38 (br. s, 1H), 2.12-2.08 (m, 6H), 1.46 (s, 9H), 1.35-1.29 (m, 6H) 13 C NMR (100 MHz, CDCl3): δ 170.5,170.2, 155.9, 99.8, 97.9, 80.1, 79.7, 79.5, 78.4, 77.4, 71.7, 69.0, 67.9, 67.5,67.4, 67.2, 57.5, 57.3, 40.4, 28.6, 21.2, 21.1, 18.3, 17.7 ESI-MS: m / z C 32 H 43 O 13 NH[M+H] + Calculated value: 566.2807; Measured value: 566.2788.

[0231] [ka] 2-O-acetyl-4-O-benzyl-3-O-methyl-α-D-rhamnopyranoside-(1→4)-2-O-acetyl-3-O-methyl-α-D-rhamnopyranoside-(1→4)-2-(tert-butoxycarbonyl)amino]ethyl 2-O-acetyl-3-O-methyl-α-D-rhamnopyranoside (S13) Compound S12 (1.46 g, 2.57 mmol) and compound S8 (1.29 g, 3.10 mmol) were coevaporated with toluene (5×20 mL) and dried under high vacuum overnight. Anhydrous methylene chloride (50 mL) was added followed by 0.400 g of activated powdered 3 Å molecular sieves. The reaction was then cooled to −78° C. under N2 atmosphere and N-iodosuccinimide (990.0 mg, 3.60 mmol) was added followed by triflic acid (0.40 mL, 4.52 mmol). The reaction was stirred for 3 hours until it reached completion. The reaction was then filtered through a Büchner funnel, diluted with CHCl (100 mL) and washed with 10% NaSO (2×100 mL) and saturated NaHCO (100 mL). The organic layer was then dried over Na2SO4, filtered, and purified by flash chromatography (eluent: EtOAc / Hexane, V / V) to give compound S13 as a clear oil (1.65 g, 1.92 mmol, 75%). Rf = 0.45 (EtOAc / Hexane, 1 / 1, V / V) [α] D 25 2.0 (c 0.46, CHCl3) 1HNMR (600 MHz, CDCl3): δ 7.37-7.26 (m, 5H),5.35 (s, 1H), 5.33 (s, 1H), 5.27 (s, 1H), 5.10 (s, 1H), 5.09 (s, 1H), 4.88 (d,1H), 4.83 (br. s, 1H), 4.61 (d, 1H), 3.85-3.71 (m, 1H), 3.77-3.65 (m, 3H), 3.59(dd, 1H), 3.56-3.45 (m, 5H), 3.44 (s, 3H), 3.40 (s, 3H), 3.38 (s, 3H),3.38-3.33 (m, 2H), 3.32-3.26 (m, 1H), 2.13 (s, 3H), 2.10 (s, 3H), 2.08 (s, 3H),1.46 (s, 9H), 1.33-1.30 (m, 6H), 1.28 (d, 3H) 13 C NMR (150 MHz, CDCl3):δ 170.4, 170.2, 170.1, 155.9, 138.6, 128.5 (2C), 128.1(2C), 127.8, 99.5, 97.9, 80.1, 80.0, 80.0, 78.7, 78.4, 75.5, 68.7, 68.5, 68.0,67.9, 67.8, 67.3, 67.1, 57.6, 57.5, 57.3, 40.4, 28.5, 21.2, 21.1, 18.3, 18.1,17.9 ESI-MS: m / z C 41 H 63 O 18 NNa [M+Na] + Calculated value, 880.3937; measured value, 880.3943.

[0232] [ka] 2-O-acetyl-3-O-methyl-α-D-rhamnopyranoside-(1→4)-2-O-acetyl-3-O-methyl-α-D-rhamnopyranoside-(1→4)-2-(tert-butoxycarbonyl)amino]ethyl 2-O-acetyl-3-O-methyl-α-D-rhamnopyranoside (S14) Palladium hydroxide on carbon (807.0 mg, 5.67 mmol) was added to MeOH (50 mL) and the solution was bubbled with H2 for 30 min. Compound S13 (1.65 g, 1.92 mmol) was then added and the solution was stirred at RT with a continuous flow of H2 for 3 h. The reaction was then filtered through a pad of Celite and evaporated to give S13 as a clear oil (1.57 g, 1.89 mmol, 98%). Rf = 0.20 (EtOAc / Hexane, 1 / 1, V / V) [α] D 25 2.3 (c 0.51, CHCl3) 1 HNMR (600 MHz, CD3OD): δ 5.40 (dd, 1H), 5.36(s, 1H), 5.32 (s, 1H), 5.10 (d, 1H), 5.06 (d, 1H), 4.75 (s, 1H), 3.88-3.82 (m,1H), 3.81-3.75 (m, 2H), 3.75-3.71 (m, 1H), 3.69 (dd, 1H), 3.57 (dd, 1H),3.53-3.45 (m, 3H), 3.43 (s, 3H), 3.40 (s, 3H), 3.39 (s, 3H), 3.35-3.32 (m, 4H),3.32-3.24 (m, 2H), 2.13-2.11 (m, 6H), 2.10 (s, 3H), 1.50 (s, 9H), 1.33-1.30 (m,6H), 1.29 (d, 3H) 13 C NMR (150 MHz, CD3OD): δ 171.7 (2C), 171.6, 158.5, 101.0, 100.8, 98.7, 81.5, 81.3, 80.8,80.4, 79.6, 72.9, 72.9, 70.8, 69.6, 69.3, 69.2, 69.0, 68.0, 67.5, 58.0, 57.7,41.1, 28.9, 20.7, 18.6, 18.5, 17.9 ESI-MS: m / z C 34 H 57 O 18 NNa[M+Na] + Calculated value, 790.3468; measured value, 790.3462.

[0233] [ka] 2-O-acetyl-4-O-benzyl-3-O-methyl-α-D-rhamnopyranoside-(1→4)-2-O-acetyl-3-O-methyl-α-D-rhamnopyranoside-(1→4)-2-O-acetyl-3-O-methyl-α-D-rhamnopyranoside-(1→4)-2-(tert-butoxycarbonyl)amino]ethyl 2-O-acetyl-3-O-methyl-α-D-rhamnopyranoside (S15) Compound S14 (1.45 g, 1.89 mmol) and compound S8 (943.9 mg, 2.27 mmol) were coevaporated with toluene (5×20 mL) and dried under high vacuum overnight. Anhydrous methylene chloride (50 mL) was added followed by 0.400 g of activated powdered 3 Å molecular sieves. The reaction was then cooled to −78° C. under N2 atmosphere and N-iodosuccinimide (727.0 mg, 2.64 mmol) was added followed by triflic acid (0.35 mL, 3.96 mmol). The reaction was stirred for 3 hours until it reached completion. The reaction was then filtered through a Büchner funnel, diluted with CHCl (100 mL) and washed with 10% NaSO (2×100 mL) and saturated NaHCO (100 mL). The organic layer was then dried over Na2SO4, filtered, and purified by flash chromatography (eluent: EtOAc / Hexane, V / V) to give compound S15 as a clear oil (1.49 g, 1.41 mmol, 74%). Rf = 0.40 (EtOAc / Hexane, 1 / 1, V / V) [α] D 25 1.7 (c 0.31, CHCl3) 1HNMR (600 MHz, CDCl3): δ 7.37-7.26 (m, 5H),5.35 (s, 3H), 5.28 (s, 1H), 5.13-5.06 (m, 3H), 4.89 (d, 1H), 4.83 (br. s, 1H),4.70 (s, 1H), 4.61 (d, 1H), 3.87-3.63 (m, 5H), 3.63-3.57 (m, 1H), 3.57-3.46 (m,7H), 3.44 (s, 3H), 3.41-3.24 (m, 13H), 2.15-2.04 (m, 12H), 1.46 (s, 9H),1.35-1.26 (m, 12H) 13 C NMR (150 MHz, CDCl3): δ 170.1, 170.3, 170.2, 170.1, 155.9, 138.6, 128.5 (2C), 128.1 (2C),127.9, 99.6, 99.5, 97.9, 80.1, 80.0, 79.9, 79.7, 78.9, 78.4, 77.4, 75.5, 68.7,68.5, 68.1, 68.0, 67.9, 67.8, 67.7, 67.3, 67.1, 40.4, 28.6, 21.3, 21.2, 18.4,18.2, 18.1, 18.0 ESI-MS: m / z C 50 H 77 NO 23 H [M+H] + Calculated value: 1060.4959; Measured value: 1060.4977.

[0234] [ka] 2-O-acetyl-3-O-methyl-α-D-rhamnopyranoside-(1→4)-2-O-acetyl-3-O-methyl-α-D-rhamnopyranoside-(1→4)-2-O-acetyl-3-O-methyl-α-D-rhamnopyranoside-(1→4)-2-(tert-butoxycarbonyl)amino]ethyl 2-O-acetyl-3-O-methyl-α-D-rhamnopyranoside (S16) Palladium hydroxide on carbon (720.0 mg, 5.13 mmol) was added to MeOH (50 mL) and the solution was bubbled with H2 for 30 min. Compound S15 (1.42 g, 1.34 mmol) was then added and the solution was stirred at RT with a continuous flow of H2 for 3 h. The reaction was then filtered through a pad of Celite and evaporated to give S16 as a clear oil (1.30 g, 1.34 mmol, 100%). Rf = 0.15 (EtOAc / Hexane, 3 / 2, V / V) [α] D 25 3.4 (c 0.67, CHCl3) 1 HNMR (600 MHz, CD3OD): δ 5.42 (dd, 1H), 5.40(dd, 1H), 5.38 (dd, 1H), 5.33 (dd, 1H), 5.10 (d, 1H), 5.08 (d, 1H), 5.06 (d,1H), 4.75 (d, 1H), 3.90-3.82 (m, 2H), 3.82-3.76 (m, 2H), 3.76-3.72 (m, 1H),3.72-3.66 (dd, 1H), 3.62-3.55 (m, 2H), 3.55-3.46 (m, 4H), 3.46-3.43 (s, 3H),3.43-3.36 (m, 11H), 3.33-3.22 (m, 2H), 2.14-2.11 (m, 9H), 2.11 (s, 3H), 1.51(s, 9H), 1.36-1.31 (m, 9H), 1.30 (d, 3H) 13 C NMR (150 MHz, CD3OD):δ 171.7 (2C), 171.6 (2C), 158.5, 101.0, 100.9, 100.8,98.7, 81.4, 81.3, 81.2, 80.9, 80.3, 80.2, 80.0, 79.6, 72.9, 70.7, 69.6, 69.3,69.2, 69.1, 69.0, 68.9, 68.0, 67.6, 58.0, 57.6, 41.1, 29.0, 20.7, 18.6, 18.5,18.4, 17.9 ESI-MS: m / z C 43 H 71 O 23 NNa [M+Na] +Calculated value: 992.4309; measured value: 992.4313.

[0235] [ka] 2-O-acetyl-4-O-benzyl-3-O-methyl-α-D-rhamnopyranoside-(1→4)-2-O-acetyl-3-O-methyl-α-D-rhamnopyranoside-(1→4)-2-O-acetyl-3-O-methyl-α-D-rhamnopyranoside-(1→4)-2-O-acetyl-3-O-methyl-α-D-rhamnopyranoside-(1→4)-2-(tert-butoxycarbonyl)amino]ethyl 2-O-acetyl-3-O-methyl-α-D-rhamnopyranoside (S17) Compound S16 (878.9 mg, 0.91 mmol) and compound S8 (452.9 mg, 1.09 mmol) were coevaporated with toluene (5×20 mL) and dried under high vacuum overnight. Anhydrous methylene chloride (50 mL) was added followed by 0.200 g of activated powdered 3 Å molecular sieves. The reaction was then cooled to −78° C. under N2 atmosphere and N-iodosuccinimide (348.8 mg, 1.27 mmol) was added followed by triflic acid (0.15 mL, 1.70 mmol). The reaction was stirred for 3 hours until completion was reached. The reaction was then filtered through a Büchner funnel, diluted with CHCl (50 mL) and washed with 10% NaSO (2×50 mL) and saturated NaHCO (50 mL). The organic layer was then dried over Na2SO4, filtered, and purified by flash chromatography (eluent: EtOAc / Hexane, V / V) to give compound S17 as a clear oil (693.0 mg, 0.55 mmol, 64%). Rf = 0.50 (EtOAc / Hexane, 3 / 2, V / V) [α] D 25 1.5 (c 0.27, CHCl3) 1HNMR (600 MHz, CDCl3): δ 7.39-7.27 (m, 5H), 5.33 (s, 4H), 5.28 (s, 1H), 5.10 (s, 4H), 4.88 (d, 1H), 4.83 (br. s, 1H), 4.70(s, 1H), 4.61 (d, 1H), 3.86-3.54 (m, 6H), 3.62-3.46 (m, 10H), 3.44 (s, 3H), 3.41-3.34(m, 13H), 3.34-3.23 (m, 2H), 2.13 (s, 3H), 2.10 (s, 3H), 2.08 (s, 9H), 1.46 (s,9H), 1.36-1.22 (m, 15H) 13 C NMR (150 MHz, CDCl3): δ 170.4, 170.3, 170.2, 170.1, 170.0, 155.9, 138.6, 128.5 (2C), 128.1(2C), 127.9, 99.7, 99.6, 99.5, 99.4, 97.9, 80.1, 80.0, 79.9, 79.7, 78.8, 78.5,78.3, 78.3, 77.4, 75.5, 68.7, 68.5, 68.0, 67.9, 67.8, 67.7, 67.3, 67.0, 57.6,57.5, 57.4 (2C), 57.3, 40.4, 28.5, 21.2, 21.1, 18.4, 18.3, 18.2, 18.1, 17.9ESI-MS: m / z C 59 H 91 O 28 NH [M+H] + Calculated value, 1262.5800; Measured value, 1262.5806.

[0236]

change

[0333] Palladium hydroxide on carbon (300.0 mg, 2.14 mmol) was added to MeOH (30 mL) and the solution was bubbled with H2 for 30 min. Compound S17 (623.3 mg, 0.49 mmol) was then added and the solution was stirred at RT with a continuous flow of H2 for 3 h. The reaction was then filtered through a pad of Celite and evaporated to give S18 as a clear oil (537.0 mg, 0.46 mmol, 93%). Rf = 0.25 (EtOAc / Hexane, 3 / 2, V / V) [α] D 25 0.12 (c 0.033, CHCl3) 1 HNMR (600 MHz, CD3OD): δ 5.44-5.41 (m, 3H),5.39 (s, 1H), 5.34 (dd, 1H), 5.11 (s, 1H), 5.09 (s, 2H), 5.07 (s, 1H), 4.76 (s,1H), 3.92-3.83 (m, 3H), 3.83-3.77 (m, 2H), 3.77-3.73 (m, 1H), 3.70 (dd, 1H),3.62-3.56 (m, 3H), 3.56-3.46 (m, 6H), 3.45 (3H), 3.45-3.38 (m, 15H), 3.34-3.24(m, 2H), 2.14-2.12 (m, 12H), 2.11 (s, 3H), 1.52 (s, 9H), 1.36-1.32 (m, 12H),1.31 (d, 3H) 13C NMR (150 MHz, CDCl3): δ 171.7 (2C), 171.6 (3C), 158.4, 101.0, 100.8, 98.6, 81.4, 81.3,81.2, 80.9, 80.3, 80.2, 81.1, 80.0, 79.6, 72.9, 70.7, 69.6, 69.3, 69.2, 69.1,69.0, 68.9, 68.0, 67.5, 58.0, 57.6, 41.1, 29.0, 20.7, 18.6, 18.5 (3C), 17.9ESI-MS: m / z C 52 H 85 O 28 NNa [M+Na] + Calculated value: 1194.5150; Measured value: 1194.5153.

[0237] [ka] 3-O-methyl-α-D-rhamnopyranoside-(1→4)-3-O-methyl-α-D-rhamnopyranoside-(1→4)-2-(tert-butoxycarbonyl)amino]ethyl 3-O-methyl-α-D-rhamnopyranoside (S19) Compound S14 (755.0 mg, 0.98 mmol) was dissolved in MeOH (30.0 mL) and sodium metal (15.0 mg, 0.65 mmol) was added. The reaction was stirred at RT for 16 h and then diluted with DowexH + The crude product was then dissolved in CH2Cl2 (100 mL) and trifluoroacetic acid (5.8 mL), stirred at RT for 10 min, and then evaporated under reduced pressure to yield S19 as a beige powder (532.4 mg, 0.98 mmol, 100%). [α] D 25 1.5 (c 0.52, CH3OH) 1H NMR (600 MHz, CD3OD): δ 5.13 (s,2H), 4.80 (s, 1H), 4.12 (s, 1H), 4.09 (s, 1H), 4.07 (s, 1H), 3.96-3.90 (m, 1H),3.80-3.74 (m, 2H), 3.67-3.51 (m, 4H), 3.52-3.45 (m, 1H), 3.46 (s, 3H), 3.44 (s,3H), 3.42 (s, 3H), 3.41-3.35 (m, 1H), 3.29-3.24 (m, 1H), 3.25-3.15 (m, 2H), 1.32 (d, 3H), 1.30 (d, 3H), 1.26 (d, 3H) 13 C NMR (150 MHz, CD3OD):δ 103.2, 103.1, 101.7, 83.1, 82.8, 82.0, 79.5, 79.3,72.6, 70.5, 69.1, 68.7, 68.3, 68.0, 67.5, 64.6, 57.3, 56.7, 56.6, 40.5, 18.7,18.6, 17.9 ESI-MS: m / z C 23 H 43 O 13 NH [M+H] + Calculated value: 542.2807; measured value: 542.2804.

[0238] [ka] 3-O-methyl-α-D-rhamnopyranoside-(1→4)-3-O-methyl-α-D-rhamnopyranoside-(1→4)-3-O-methyl-α-D-rhamnopyranoside-(1→4)-2-(tert-butoxycarbonyl)amino]ethyl 3-O-methyl-α-D-rhamnopyranoside (S20) Compound S16 (390 mg, 0.40 mmol) was dissolved in MeOH (30.0 mL) and sodium metal (10 mg, 0.44 mmol) was added. The reaction was stirred at RT for 16 h and then diluted with DowexH +The crude product was then dissolved in CH2Cl2 (50 mL) and trifluoroacetic acid (2.4 mL), stirred at RT for 10 min, and then evaporated under reduced pressure to yield S20 as a beige powder (282.1 mg, 0.40 mmol, 100%). [α] D 25 1.1 (c 0.13, CH3OH) 1 H NMR (600 MHz, CD3OD): δ 5.14 (m,3H), 4.81 (s, 1H), 4.13 (s, 1H), 4.09 (s, 3H), 3.96-3.91 (m, 1H), 3.82-3.75 (m,2H), 3.75-3.67 (m, 2H), 3.67-3.57 (m, 4H), 3.57-3.52 (m, 1H), 3.51-3.42 (m,12H), 3.42-3.36 (m, 3H), 3.31-3.26 (m, 1H), 3.25-3.15 (m, 2H), 1.34 (d, 3H),1.32-1.28 (m, 6H), 1.27 (d, 3H) 13 C NMR (150 MHz, CD3OD):δ 103.2, 103.1, 103.0, 101.7, 83.1, 83.0, 82.8, 79.4,79.3, 79.2, 72.6, 70.5, 69.1, 68.7, 68.4, 68.4, 68.0, 67.5, 64.6, 57.3, 56.7,56.6, 40.4, 18.7, 18.5 (2C), 17.9 ESI-MS: m / z C 30 H 55 O 17 NH[M+H] + Calculated value, 702.3543; measured value, 702.3532.

[0239] [ka] 3-O-methyl-α-D-rhamnopyranoside-(1→4)-3-O-methyl-α-D-rhamnopyranoside-(1→4)-3-O-methyl-α-D-rhamnopyranoside-(1→4)-3-O-methyl-α-D-rhamnopyranoside-(1→4)-2-(tert-butoxycarbonyl)amino]ethyl 3-O-methyl-α-D-rhamnopyranoside (S21) Compound S18 (500 mg, 0.47 mmol) was dissolved in MeOH (30.0 mL) and sodium metal (10 mg, 0.44 mmol) was added. The reaction was stirred at RT for 16 h and then diluted with DowexH + The crude product was then dissolved in CH2Cl2 (50 mL) and trifluoroacetic acid (2.7 mL), stirred at RT for 10 min, and then evaporated under reduced pressure to give S21 as a beige powder (402 mg, 0.47 mmol, 100%). [α] D 25 1.0 (c 0.26, CH3OH) 1 H NMR (600 MHz, CD3OD): δ 5.15 (s,4H), 4.80 (s, 1H), 4.14-4.12 (m, 1H), 4.12-4.08 (m, 4H), 3.96-3.91 (m, 1H), 3.81-3.74(m, 3H), 3.75-3.67 (m, 2H), 3.67-3.58 (5H), 3.57-3.52 (dd, 1H), 3.50-3.38 (m,19H), 3.31-3.28 (dd, 1H), 3.26-3.15 (m, 2H), 1.34 (d, 3H), 1.32-1.28 (m, 9H),1.27 (d, 3H) 13 C NMR (150 MHz, CD3OD): δ 103.2, 103.1, 103.0 (2C), 101.7, 83.1 (3C), 82.8, 82.0, 79.4, 79.3,79.2 (2C), 72.6, 70.5, 69.1 (2C), 68.7, 68.4, 67.4 (2C), 67.5, 64.6, 57.3,56.7, 56.6 (2C), 40.4, 18.7, 18.6 (3C), 17.9 ESI-MS: m / z C 37 H67 O 21 NH[M+H] + Calculated value: 862.4278; Measured value: 862.4265.

[0240] [ka] 3-O-methyl-α-D-rhamnopyranoside-(1→4)-3-O-methyl-α-D-rhamnopyranoside-(1→4)-2-(2,2-dimethoxybutylcarbonyl)amino]ethyl 3-O-methyl-α-D-rhamnopyranoside (S22) 2,5-Dioxopyrrolidin-1-yl 5,5-dimethoxypentanoic acid (239 mg, 0.92 mmol) was dissolved in anhydrous DMF (9.2 mL) and compound S19 (50 mg, 92 μmol) was added to this solution. The reaction was stirred at RT for 16 h and driven to completion by adding one drop of Et3N. The sample was evaporated under reduced pressure and coevaporated with toluene (5×10 mL). The crude sample was then suspended in HO (2 mL) and extracted with CHCl3 (5×1 mL). The aqueous layer was collected, evaporated, and purified by HPLC (C-18, HO / MeOH) to yield S22 as a white powder (7.4 mg, 10.8 μmol, 12%). 1 H NMR (600 MHz, CD3OD): δ 5.16-5.13 (m, 2H), 4.75 (d, 1H), 4.41 (t, 0.73H), 4.13-4.08 (m,2H), 4.08-4.06 (m, 1H), 3.84-3.71 (m, 3.5H), 3.71-3.65 (m, 1H), 3.64-3.55 (m,3H), 3.55-3.37 (m, 17H), 3.36-3.32 (m, 4H), 3.29 (dd, 1H), 2.26 (t, 1.6H),1.73-1.60 (m, 3.3H), 1.33-1.25 (m, 9H) 13C NMR (150 MHz, CD3OD):δ 176.2, 105.8, 103.2, 103.1, 101.3, 83.2, 83.1, 83.0,82.0, 79.7, 79.2, 72.6, 70.5, 69.0, 68.3, 68.0, 67.6, 67.0, 57.3, 56.7, 53.5,53.4, 40.2, 36.6, 33.1, 22.0, 18.7, 18.6, 17.8.

[0241] [ka] 3-O-methyl-α-D-rhamnopyranoside-(1→4)-3-O-methyl-α-D-rhamnopyranoside-(1→4)-3-O-methyl-α-D-rhamnopyranoside-(1→4)-2-(2,2-dimethoxybutylcarbonyl)amino]ethyl 3-O-methyl-α-D-rhamnopyranoside (S23) 2,5-Dioxopyrrolidin-1-yl 5,5-dimethoxypentanoic acid (185 mg, 0.71 mmol) was dissolved in anhydrous DMF (7.1 mL) and compound S20 (50.0 mg, 71.2 μmol) was added to this solution. The reaction was stirred at RT for 16 h and driven to completion by adding one drop of Et3N. The sample was evaporated under reduced pressure and co-evaporated with toluene (5×10 mL). The crude sample was then suspended in HO (2 mL) and extracted with CHCl3 (5×1 mL). The aqueous layer was collected, evaporated and purified by HPLC (C-18, eluent: HO / MeOH, V / V) to yield S23 as a white powder (11.0 mg, 13.0 μmol, 18%). 1H NMR (600 MHz, CD3OD): δ 5.16-5.13 (m, 3H), 4.75 (d, 1H), 4.41 (t, 1H), 4.13-4.09 (m, 3H),4.07 (dd, 1H), 3.82-3.71 (m, 4H), 3.71-3.65 (m, 1H), 3.64-3.56 (m, 3H),3.54-3.47 (m, 6H), 3.47-3.36 (m, 14H), 3.35-3.32 (m, 6H), 3.31 (dd, 1H), 2.26(t, 2H), 1.72-1.61 (m, 4H), 1.34-1.25 (m, 12H) 13 C NMR (150 MHz, CD3OD):δ 176.0, 105.8, 103.2, 103.1, 103.0, 101.3, 83.1, 83.0,82.9, 82.0, 79.8, 79.4, 79.2, 72.6, 70.5, 69.1, 69.0, 68.4, 68.3, 68.0, 67.7,67.0, 57.3, 56.7, 53.5, 53.4, 40.2, 36.6, 33.1, 22.0, 18.7, 18.6 (2C), 17.9.

[0242] [ka] 3-O-methyl-α-D-rhamnopyranoside-(1→4)-3-O-methyl-α-D-rhamnopyranoside-(1→4)-3-O-methyl-α-D-rhamnopyranoside-(1→4)-3-O-methyl-α-D-rhamnopyranoside-(1→4)-2-(2,2-dimethoxybutylcarbonyl)amino]ethyl 3-O-methyl-α-D-rhamnopyranoside (S24) 2,5-Dioxopyrrolidin-1-yl 5,5-dimethoxypentanoic acid (150 mg, 0.58 mmol) was dissolved in anhydrous DMF (5.8 mL) and compound S21 (50 mg, 58.0 μmol) was added to this solution. The reaction was stirred at RT for 16 h and driven to completion by adding one drop of Et3N. The sample was evaporated under reduced pressure and co-evaporated with toluene (5×10 mL). The crude sample was then suspended in HO (2 mL) and extracted with CHCl3 (5×1 mL). The aqueous layer was collected, evaporated, and purified by HPLC (C-18, eluent: HO / MeOH) to yield S24 as a white powder (20.3 mg, 20.2 μmol, 35%). 1 H NMR (600 MHz, CD3OD): δ 5.17-5.13 (m, 4H), 4.75 (d, 1H), 4.41 (t, 1H), 4.13-4.10 (m, 4H),4.07 (dd, 1H), 3.82-3.71 (m, 5H), 3.71-3.65 (m, 1H), 3.65-3.56 (m, 4H),3.52-3.38 (m, 22H), 3.36-3.33 (m, 7H), 3.31 (dd, 1H), 2.26 (t, 1H), 1.73-1.61(m, 4H), 1.34-1.25 (m, 15H) 13 C NMR (150 MHz, CD3OD): δ 176.0, 105.8, 103.2, 103.1, 101.3, 83.1, 83.0, 82.0, 79.8, 79.4,79.2, 72.7, 70.5, 69.0 (2C), 68.3, 68.0 (2C), 67.6, 67.1, 57.3, 56.7, 53.5,53.4, 40.2, 36.6, 33.1, 22.0, 18.7, 18.6 (3C), 17.9.

[0243] Example 6: Screening of new oligosaccharide linkers against mAb 1B1 by inhibition ELISA Linker-linked oligosaccharides (Example 5) are shown to efficiently mimic the methyl rhamnan chip epitope. 1B1 mAb, previously identified as specific for methyl rhamnan chip, at a constant concentration of 10 μg / ml in PBS-Tween was incubated with dilutions of Pseudomonas aeruginosa (Pa) PAO1 BAA-47(wt) lipopolysaccharide (LPS) (positive control for inhibition) and Neisseria meningitidis (Nm) galE / lpt3 LPS (negative control for inhibition) and a synthetic oligosaccharide with a linker (S19-21) in a 1:1 ratio. The final concentration of mAb 1B1 was 5 μg / ml. Pa LPS and Nm LPS and synthetic oligosaccharides were titrated starting at a concentration of 3 mg / ml (final concentration 1.5 mg / ml) and 12 two-fold dilutions were performed in PBS-Tween. The mixtures were incubated together for 1 h at room temperature before being added to Pa wtLPS-coated ELISA plates for 1 h at room temperature.

[0244] If there is enough LPS / oligosaccharide present to bind all of the available 1B1 in the inhibition step, there will be no free 1B1 to bind to the LPS on the ELISA plate and therefore no color reaction by the ELISA, but if none of the LPS / oligosaccharide binds to the antibody or there is not enough LPS / oligosaccharide present to block 1B1 binding, the intensity of the color produced by the ELISA will be similar to that of incubation with PBS alone.

[0245] As shown in Figure 33, Pa wtLPS binds and blocks 1B1 as no color is produced by ELISA until the LPS is diluted to approximately 100 μg / ml. Irrelevant Nm LPS does not block 1B1 binding, nor does PBS.

[0246] Oligosaccharides block the binding of 1B1 to tetra- and pentasaccharides and behave similarly with small titers of approximately 5 μg / ml measured, whereas trisaccharides also block binding but titers of approximately 100 μg / ml are measured earlier.

[0247] These results therefore support the data of the previously prepared oligosaccharides (without linker). The addition of the linker in these oligosaccharides does not alter the conformation of the oligosaccharides, nor does it affect the binding and blocking of the oligosaccharides to mAb 1B1. Thus, the synthetic oligosaccharides with linkers effectively mimic the epitope recognized by mAb 1B1 on Pa.

[0248] Example 7: Conjugation of oligosaccharide linkers with activated CRM (for immunization) and activated BSA (for screening) for the preparation of glycoconjugates Aminooxy activation of CRM and BSA: First, the lysin of the protein (CRM or BSA) was activated by dissolving it in 200 mM sodium phosphate buffer pH 7.4 at 10 mg / ml and cooling to 4° C. Then, approximately 85× molar excess of bromoacetic acid N-hydroxysuccinimide ester dissolved in DMSO at 3 mg / ml was added and left at 4° C. for 18 hours. The bromine-activated protein was then desalted (3 times) against water using an amicon ultra-10 30K MWCO spin column to a volume of approximately 500 μl. To this was added 500 μl of 200 mM sodium phosphate buffer pH 7.4 and cooled to 4° C. Approximately 75× molar excess of 3-(aminooxy)-1-propanethiol hydrochloride dissolved in 100 μl of 200 mM sodium phosphate buffer pH 7.4 was added and left at room temperature for 2 hours. The resulting aminooxy-activated protein was then desalted against water (three times) using an amicon ultra-10 30K MWCO spin column. Activation levels were determined by MALDI MS (Table 6).

[0249] Activation of the linkers to generate aldehyde functional groups: To convert the linkers on S22, S23, and S24 to active aldehyde functional groups, the oligosaccharides (S22-24) were dissolved in 50% acetic acid at 3 mg / ml and left at 37° C. for 7 h. Once cooled, the reaction mixture was lyophilized.

[0250] Conjugation and characterization of the conjugates: 1 mg of activated oligosaccharide (tri-, tetra- or penta-) dissolved in 50 μl of 200 mM sodium phosphate buffer pH 6 was added to the activated protein at 0, 3 and 6 hours. The amount of activated protein used was adjusted in each case to maintain approximately 8× molar excess of oligosaccharide per aminooxy group of protein. The reaction was left at room temperature for 18 hours. The product was then isolated (3 times) against PBS using amicon ultra-10 30K MWCO spin columns and stored at 4° C. Activated oligosaccharides (tri-, tetra-, and penta-) were conjugated at approximately 8x molar ratio per aminooxy group on the activated CRM (tri- / tetra- / penta- are 2.1 mg / 1.7 mg / 1.5 mg CRM-oxy, respectively). The degree of conjugation was determined by MALDI MS (Table 6).

[0251] [Table 6]

[0252] Example 8: Immunization of mice and rabbits with the conjugates Female BALB / c mice aged 6-8 weeks were immunized intraperitoneally three times. At each time point, each mouse received the same amount of oligosaccharide conjugate as well as SIGMA adjuvant and PBS buffer. Mice were primed on day 0 and boosted on days 21 and 42, and blood samples were taken on days 0, 35, and 56.

[0253] Each mouse in the MRha3V group was administered 3 μg of trisaccharide conjugate, yielding 28 μg of CRM, with 50% v / v SIGMA adjuvant, and PBS buffer was administered intraperitoneally in a total volume of 100 μl. Each mouse in the MRha4V group was administered 3 μg of tetrasaccharide conjugate, yielding 25.5 μg of CRM, with 50% v / v SIGMA adjuvant, and PBS buffer was administered intraperitoneally in a total volume of 100 μl. Finally, each mouse in the MRha5V group was administered 3 μg of pentasaccharide, yielding 23 μg of CRM, with 50% v / v SIGMA adjuvant, and PBS buffer was administered intraperitoneally in a total volume of 100 μl. Blood samples were obtained by submandibular vein collection method, and approximately 100 μl of serum was obtained after blood separation.

[0254] Example 9: Screening of generated mouse sera against BSA-conjugates and LPS Individual sera from mice subjected to a prime-one-boost-two immunization regimen were screened by ELISA for the ability to recognize BSA-oligosaccharide conjugates and Pa wtLPS. All mice developed good IgM responses to the conjugates, as exemplified by recognition of BSA-oligosaccharide conjugates by ELISA (FIGS. 35A-C) compared to pre-immune serum (FIG. 34A) and moderate responses to LPS (FIG. 35D) compared to pre-immune serum (FIG. 34B). All mice developed moderate IgG responses to the conjugates, as exemplified by recognition of BSA-oligosaccharide conjugates by ELISA (FIG. 37A) compared to pre-immune serum (FIG. 36A). Similarly, all mice administered the tetrasaccharide and pentasaccharide conjugates developed moderate IgG responses to the conjugates, as exemplified by recognition of LPS by ELISA (FIG. 37B) compared to pre-immune serum (FIG. 36B). The mouse data may suggest that the minimum length oligosaccharide required for efficient mimicry of the native antigen is a tetrasaccharide, as mice immunized with CRM-tetrasaccharide and CRM-pentasaccharide conjugates showed enhanced IgG responses to Pa wtLPS by ELISA compared to mice receiving the trisaccharide conjugate (Figure 37B). Killed whole cells detailed in Table 7 were screened for recognition by generated sera by ELISA.

[0255] Example 10: Screening of Rabbit Serum Produced Against BSA-Conjugates and LPS All rabbits generated a good immune response to the conjugate, as exemplified by the recognition of the BSA-oligosaccharide conjugate by ELISA compared to pre-immune serum (Figure 38A). Similarly, all rabbits generated a good immune response to the conjugate, as exemplified by the recognition of LPS by ELISA compared to pre-immune serum (Figure 38B). All rabbits generated a strong response (endpoint titers in the range of 1:10,000), which allowed the recognition of several different LPS molecules.

[0256] Example 11: Screening of generated mouse and rabbit sera against sterile whole cells Whole-cell ELISAs were performed against a variety of P. aeruginosa bactericidal cells (Table 7 ), including wild-type strains, strains with mutations in genes thought to be associated with A-band methylrhamnan, and serotype strains most commonly encountered in the clinical setting.

[0257] [Table 7]

[0258] Post-immunization mouse sera pooled with the administered oligosaccharide vaccine recognized the cells as shown in Figure 39 A. Again, post-immunization sera with the trisaccharide conjugate induced a weak response compared to the clear responses induced by the tetrasaccharide and pentasaccharide conjugates, illustrating that the methylrhamnan chip epitope is visible in the context of whole cells and that the oligosaccharide conjugates are capable of generating an immune response capable of recognizing this epitope.

[0259] Individual rabbit sera after immunization recognized cells as shown in Figure 39B, thereby making the methylrhamnan chip epitope visible in the context of the whole cell, illustrating that oligosaccharide conjugates (tri-, tetra-, and penta-) are capable of eliciting an immune response capable of recognizing this epitope. Moreover, when rabbit sera were screened against various clinical isolates from our collection (Table 7) (Figure 39C), again good cross-reactivity was observed against most strains, further illustrating the conservation of this epitope and the ability of oligosaccharide-based conjugates to elicit the required immune response.

[0260] In agreement with previous data with LPS and BSA conjugates, whole cell ELISA confirmed the requirement in mice for the tetrasaccharide as the minimal length oligosaccharide required to stimulate an adequate response, however, serum from rabbits did not show this same minimum length requirement as all conjugates were able to induce similar cross-reactive responses.

[0261] Taken together, these results demonstrate that at least synthetic tetra- and pentasaccharide CRM conjugates representing methylrhamnan A band CHIP epitopes can elicit specific immune responses that recognize Pa wtLPS and whole cells representative of the serotypes most commonly encountered in the clinical setting, thereby illustrating their potential as a viable alternative to isolated antigens as vaccine immunogens.

[0262] The specific embodiments described herein are intended to be examples only. Modifications, modifications, and variations may be made to the specific embodiments by those skilled in the art. The claims should not be limited by the specific embodiments described herein, but should be interpreted consistent with the specification as a whole.

[0263] All publications, patents, and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains, and are hereby incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0371] The invention being thus described, it will be apparent that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the invention, and all such modifications, as would be apparent to one skilled in the art, are intended to be included within the scope of the following claims.

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Makarenko et al., Immunological studiesof an artificial antigen with specificity of a common polysaccharide antigen ofPseudomonas aeruginosa. FEMS Imm. Med. Microbiol. (1993) 7:251-256. 26. Lam et al., Occurrence of a commonlipopolysaccharide antigen in standard and clinical strains of Pseudomonasaeruginosa. (1989) J. Clin. Microbiol. 27:962-967. 27. Richard et al., Antibody binding to theO-specific antigen of Pseudomonas aeruginosa O6 inhibits cell growth. Antimrob.Ag. Chemother. (2020) 64: e02168-19 28. Zunk, M. and Kiefel, M. J., Anefficient synthesis of selectively functionalized d-rhamnose derivatives,Tetrahedron Lett., 52 (2011) 1296-1299. 29. Faure et al., Practical synthesis ofvaluable d-rhamnoside building blocks for oligosaccharide synthesis, Tetrahedron.Lett. 48 (2007) 2385-2388. 30. Seepersaud et al., Ceric ammoniumnitrate / acetic anhydride: A tunable system for the O-acetylation andmononitration of diversely protected carbohydates, Synth. Commun. 47 (2017)853-871. 31. 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Dass, C., Fundamentals of ContemporaryMass Spectrometry, Hoboken, New Jersey, John Wiley & Sons, Inc., 2007, pp.17-26. 37. Crawford, C. J. and Oscarson, S.,Convergent total synthesis of Cryptococcus neoformans serotype B capsulerepeating motif, Carbohydr. Res. 497 (2020), https: / / doi.org / 10.1016 / j.carres. 38. Qin et al., Chemical synthesis ofPseudomonas aeruginosa O11 O-antigen trisaccharide based on neighboringelectron-donating effect, J. Carbohydr. Chem. 39 (2020) 374-397. 39. X. Zhang, D. Wang, G. Jin, L. Wang, Z.Guo, G. Gu, Synthesis of a tetrasaccharide repeating unit of theexopolysaccharide from Burkholderia multivorans, J. Carb. Chem. 36 (2017)189-204. 40. D. Wang, W. Zhuge, Z. Guo, G. Gu,Synthesis of a disaccharide repeating unit of the O-antigen from Burkholderiaambifaria and its oligomers, Carb. Res. 442 (2017) 41-51. 41. M. P. Jamshidi, C. Cairns, S. Chong, F.St. Micheal, E. V. Vinogradov, A. D. Cox, J. 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Claims

1. Formula A: α-Rha3OMe (-4α-Rha3OMe) ｎ - Formula A an antigenic compound comprising an oligosaccharide moiety of In the formula, n is 1 to 5, preferably 2 to 4, An antigenic compound, wherein two positions of each Rha3OMe sugar moiety are independently substituted with -OAc or -OH.

2. Formula A1: α-Rha3OMe (-4α-Rha3OMe) ｎ -X Formula A1 and wherein n is 1 to 5; X is -H or -(4α-Man3OMe) ｍ - a handle, m is 0, 1 or 2, preferably 0 or 1; 2. The antigenic compound of claim 1, wherein two positions of each Rha3OMe sugar moiety are independently substituted with -OAc or -OH.

3. When m is 1 or 2, the handle is 2-glyceraldehyde, and when m is 0, the handle is -(CH2) ｚ NH ２ 3. The antigenic compound of claim 2, wherein z is an integer selected from the group consisting of 1 to 5.

4. α-D-Rha3OMe-4-(α-D-Rha3OMe-4) ４ -4-α-D-Man3OMe-2-glyceraldehyde-1d (OS2), 3-O-methyl-α-D-rhamnopyranoside-(1→4)-3-O-methyl-α-D-rhamnopyranoside-(1→4)-3-O-methyl-α-D-rhamnopyranoside-(1→4)-3-O-methyl-α-D-rhamnopyranose (pentasaccharide), 3-O-methyl-α-D-rhamnopyranoside-(1→4)-3-O-methyl-α-D-rhamnopyranoside-(1→4)-3-O-methyl-α-D-rhamnopyranose (tetrasaccharide), 3-O-methyl-α-D-rhamnopyranoside-(1→4)-3-O-methyl-α-D-rhamnopyranoside-(1→4)-3-O-methyl-α-D-rhamnopyranose (trisaccharide) 【Chemistry 1】 2. The antigenic compound of claim 1, selected from the group consisting of:

5. Further comprising a linker for attachment to a carrier protein, the linker having the formula A2: α-Rha3OMe (-4α-Rha3OMe) ｎ -X-Linker Formula A2 and wherein n is 1 to 5; X is -(4α-Man3OMe) ｍ -(handle) ｐ - and m is 0, 1 or 2, preferably 0 or 1; p is 0 or 1; 2. The antigenic compound of claim 1, wherein two positions of each Rha3OMe sugar moiety are independently substituted with -OAc or -OH.

6. A conjugate comprising the antigenic compound of any one of claims 1 to 5 conjugated to a carrier protein.

7. 7. The conjugate of claim 6, wherein the carrier protein comprises CRM197, tetanus toxoid (TT), Pseudomonas aeruginosa protein, human serum albumin (HSA), bovine serum albumin (BSA), diphtheria toxin B fragment (DTFB), DTFB C8, diphtheria toxoid (DT), TT C fragment, pertussis toxoid, cholera toxoid, E. coli LT, E. coli ST, or exotoxin A from Pseudomonas aeruginosa.

8. A conjugate comprising an antigenic compound according to any one of claims 1 to 5 or said antigenic compound conjugated to a carrier protein, and Pharmaceutically acceptable diluents, adjuvants, carriers or excipients 10. A pharmaceutical composition comprising:

9. A vaccine comprising the antigenic compound according to any one of claims 1 to 5, a conjugate comprising said antigenic compound conjugated to a carrier protein, or the pharmaceutical composition according to claim 8.

10. 10. A composition for use in generating an immune response in a subject, the composition comprising an antigenic compound according to any one of claims 1 to 5, a conjugate comprising said antigenic compound conjugated to a carrier protein, or a pharmaceutical composition or vaccine comprising said antigenic compound or conjugate.

11. An antigenic compound according to any one of claims 1 to 5. a conjugate comprising the antigenic compound conjugated to a carrier protein; Pseudomonas aeruginosa LPS, Pseudomonas aeruginosa cells, and / or formula: α-D-Rha3OMe-4-(α-D-Rha3OMe-4) 4-4-α-D-Man3OMe-2-glyceraldehyde-1d (OS2) Isolated oxidized A-band terminal epitope antigen (OS2) An antibody or antigen-binding fragment thereof that selectively binds to

12. The antibody or antigen-binding fragment thereof of claim 11, which is a monoclonal antibody or antigen-binding fragment thereof.

13. The antibody or antigen-binding fragment thereof of claim 11, which is a chimeric or humanized antibody.

14. a heavy chain variable domain comprising a variable heavy chain CDR1, a variable heavy chain CDR2, and a variable heavy chain CDR3; the variable heavy chain CDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:10, and SEQ ID NO:19; the variable heavy chain CDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:11, and SEQ ID NO:20; The antibody or antigen-binding fragment thereof of claim 11, wherein the variable heavy chain CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 12, and SEQ ID NO:

21.

15. a light chain variable domain comprising a variable light chain CDR1, a variable light chain CDR2, and a variable light chain CDR3; the variable light chain CDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:15, and SEQ ID NO:24; the variable light chain CDR2 comprises an amino acid sequence selected from the group consisting of GTS and RVS; The antibody or antigen-binding fragment thereof of claim 14, wherein the variable light chain CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:16, and SEQ ID NO:

25.

16. a combination of a heavy chain variable domain (VH) and a light chain variable domain (VL), said combination comprising: VH comprising the amino acid sequence of SEQ ID NO: 4 and VL comprising the amino acid sequence of SEQ ID NO: 8; VH comprising the amino acid sequence of SEQ ID NO: 13 and VL comprising the amino acid sequence of SEQ ID NO: 17; and VH comprising the amino acid sequence of SEQ ID NO: 22 and VL comprising the amino acid sequence of SEQ ID NO: 26 The antibody or antigen-binding fragment thereof of claim 11, selected from the group consisting of:

17. The antibody or antigen-binding fragment thereof of claim 11 for use in the treatment of a Pseudomonas aeruginosa infection.

18. The antibody or antigen-binding fragment thereof of claim 11 for use in diagnosing Pseudomonas aeruginosa infection.

19. 12. A method for the diagnosis of Pseudomonas aeruginosa bacterial infection in an animal, preferably a human, comprising contacting a test sample with the antibody or antigen-binding fragment thereof of claim 11 and detecting specific binding thereto.

20. 3. A synthetic process for producing an antigenic compound of formula A1 according to claim 2, wherein m is 0, comprising: anomeric deprotection of the 3-O-methylated rhamnopyranoside to form a 3-O-methylated rhamnopyranose; acetylating the 3-O-methylated rhamnopyranose to form an acetylated 3-O-methylated rhamnopyranoside; partially deprotecting O-4 of the acetylated 3-O-methylated rhamnopyranoside to form a deprotected acetylated 3-O-methylated rhamnopyranoside; coupling the O-4 deprotected acetylated 3-O-methylated rhamnopyranoside to form an acetylated 3-O-methylated oligosaccharide; deprotecting the O-4 of the acetylated 3-O-methylated oligosaccharide to form an O-4 deprotected acetylated 3-O-methylated oligosaccharide; deacetylating the partially deprotected acetylated 3-O-methylated oligosaccharide to form an antigenic compound of formula A1; The process includes:

21. 3. A synthetic process for producing an antigenic compound of formula A1 according to claim 2, wherein X is a handle, comprising: glycosylation of an activated O-3 methylated rhamnopyranoside intermediate having a handle containing a protected amine at 1-O to form a protected 1-O glycoside intermediate, the activated monorhamnopyranoside intermediate containing a protecting group at the glycosylation site 4-O; removing the protecting group from 4-O to form a deprotected 1-O glycoside intermediate; coupling the deprotected 1-O glycoside intermediate to an activated O-3 methylated rhamnopyranoside intermediate, wherein the activated rhamnopyranoside intermediate contains a protecting group at 4-O, and forming a protected methylated disaccharide, trisaccharide, tetrasaccharide, or pentasaccharide; and removing all protecting groups from the protected disaccharide, trisaccharide, tetrasaccharide, or pentasaccharide. The process includes:

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