STABLE VACCINE AGAINST CLOSTRIDIUM DIFFICILE

Abstract

The present invention relates to a synthetic saccharide of general formulate (I) that is related to Clostridium difficile PS-II cell-surface polysaccharide and conjugate thereof. Said synthetic saccharide, said conjugate and pharmaceutical composition containing said synthetic saccharide or said conjugate are useful for prevention and/or treatment of diseases associated with Clostridium difficile. Furthermore, the synthetic saccharide of general formula (I) is useful as marker in immunological assays for detection of antibodies against Clostridium difficile bacteria.

Claims

1. A saccharide of general formula (I) ##STR00402## wherein n is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10; T*- represents H—, —P(═O)(OH).sub.2, —P(═O)(O.sup.−)(OH) or —PO.sub.3.sup.2−; Z represents ##STR00403## L represents a linker and; E represents —NH.sub.2, —N.sub.3, —CN, —O—NH.sub.2, —CH═CH.sub.2, —C≡CH, —Br, —Cl, —I, —CO.sub.2R′, —CONH—NH.sub.2, —SH, —OH or —SAc; R′ represents —H, -Me, -Et, 4-nitrophenyl, pentafluorophenyl, or N-succinimidyl; or a diastereoisomer or a pharmaceutically acceptable salt thereof.

2. The saccharide according to claim 1, wherein T*- represents —P(═O)(OH).sub.2, —P(═O)(O.sup.−)(OH) or —PO.sub.3.sup.2−, or a pharmaceutically acceptable salt thereof.

3. The saccharide according to claim 1, wherein Z represents ##STR00404## or a pharmaceutically acceptable salt thereof.

4. The saccharide according to claim 1, wherein -L- represents -L.sup.a-, -L.sup.a-L.sup.e-, -L.sup.a-L.sup.b-L.sup.e-, or -L.sup.a-L.sup.d-L.sup.e-; -L.sup.a- represents —(CH.sub.2).sub.o—, —(CH.sub.2—CH.sub.2—O).sub.o—C.sub.2H.sub.4—, or —(CH.sub.2—CH.sub.2—O).sub.o—CH.sub.2; -L.sup.b- represents —O—; -L.sup.d- represents —(CH.sub.2).sub.q—, —(CH(OH)).sub.q—, —(CF.sub.2).sub.q—, —(CH.sub.2—CH.sub.2—O).sub.q—C.sub.2H.sub.4—, or —(CH.sub.2—CH.sub.2—O).sub.q—CH.sub.2—; -L.sup.e-represents —(CH.sub.2).sub.p1—, —(CF.sub.2).sub.p1—, —C.sub.2H.sub.4—(O—CH.sub.2—CH.sub.2).sub.p1—, —CH.sub.2—(O—CH.sub.2—CH.sub.2).sub.p1— or —(CH.sub.2).sub.p1—O—(CH.sub.2).sub.p2—; and o, q, p1 and p2 are independently of each other an integer selected from 1, 2, 3, 4, 5, and 6, or a pharmaceutically acceptable salt thereof.

5. The saccharide according to claim 1 selected from the group consisting of: ##STR00405## ##STR00406## ##STR00407## ##STR00408## ##STR00409## ##STR00410## ##STR00411## ##STR00412## ##STR00413## ##STR00414## ##STR00415## ##STR00416## ##STR00417## ##STR00418## ##STR00419## ##STR00420## wherein Z represents ##STR00421## or a pharmaceutically acceptable salt thereof.

6. The saccharide according to claim 5 of formula (I′a-4) or formula (I′b-4), ##STR00422## wherein Z represents ##STR00423## or a pharmaceutically acceptable salt thereof.

7. A conjugate comprising a saccharide according to claim 1 covalently linked to an immunogenic carrier through the residue E of the —O-L-E group, or a pharmaceutically acceptable salt thereof.

8. The conjugate according to claim 7 of general formula (IV) ##STR00424## wherein c is comprised between 2 and 18; -E.sub.1- represents a covalent bond, —NH—, —O—NH—, —O—, —S—, —CO—, —CH═CH—, —CONH—, —CO—NHNH—, ##STR00425## —W— is selected from: ##STR00426## a represents an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10, b represents an integer selected from 1, 2, 3 and 4, CP is a carrier protein; and n is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10; T*- represents H—, —P(═O)(OH).sub.2, —P(═O)(O.sup.−)(OH) or —PO.sub.3.sup.2−; Z represents ##STR00427## L represents a linker, or a pharmaceutically acceptable salt thereof.

9. The conjugate according to claim 8, wherein the conjugate has any one of the following formulae (IV-1)-(IV-4) ##STR00428## wherein L, E.sub.1, W, c, CP, and n have the same meanings as defined in claim 8, or a pharmaceutically acceptable salt thereof.

10. The conjugate according to claim 8 having the following formula (V-2) ##STR00429## wherein L is —(CH.sub.2).sub.5—, E.sub.1 is —NH—, n is an integer selected from 1 or 2, c and W have the meaning as defined in claim 8, or a pharmaceutically acceptable salt thereof.

11. The conjugate according to claim 10, wherein —W— is ##STR00430## and a represents an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10, or a pharmaceutically acceptable salt thereof.

12. (canceled)

13. (canceled)

14. (canceled)

15. A method for detecting antibodies against bacteria containing in their cell-wall polysaccharide one of the following saccharide fragments: -6)-β-D-Glc-(1, 3)-β-D-GalNAc-(1, 4)-α-D-Glc-(1, 4)-[β-D-Glc-(1, 3)]-β-D-GalNAc-(1, 3)-α-D-Man-(1-; -3)-α-D-Man-(1, 6)-β-D-Glc-(1, 3)-β-D-GalNAc-(1, 4)-α-D-Glc-(1, 4)-[β-D-Glc-(1, 3)]-β-D-GalNAc-(1; -4)-[β-D-Glc-(1, 3)]-β-D-GalNAc-(1, 3)-α-D-Man-(1, 6)-β-D-Glc-(1, 3)-β-D-GalNAc-(1, 4)-α-D-Glc-(1; -4)-α-D-Glc-(1, 4)-[β-D-Glc-(1, 3)]-β-D-GalNAc-(1, 3)-α-D-Man-(1, 6)-β-D-Glc-(1, 3)-β-D-GalNAc-(1; and -3)-β-D-GalNAc-(1, 4)-α-D-Glc-(1, 4)-[β-D-Glc-(1, 3)]-β-D-GalNAc-(1, 3)-α-D-Man-(1, 6)-β-D-Glc-(1, the method comprising use of a saccharide according to claim 1, or a pharmaceutically acceptable salt thereof, as a marker.

16. A method for synthesis of saccharide of general formula (I) comprising of: E1) Providing a monosaccharide of formula 52*: ##STR00431##  wherein P.sup.1, P.sup.3, P.sup.4 and P.sup.25 represent protecting groups; and E2) reacting monosaccharide of formula 52* with compound of formula 2* to obtain compound 53*: ##STR00432##  wherein P.sup.1, P.sup.3, P.sup.4-P.sup.10 and P.sup.25 represent protecting groups, LG.sup.2 represents a leaving group and N.sub.p represents a protected amino group; and E3) Performing removal of protecting group P.sup.5 of compound 53* to obtain compound 54* ##STR00433##  wherein P.sup.1, P.sup.3, P.sup.4, P.sup.6-P.sup.10 and P.sup.25 represent protecting groups, and N.sub.p represents a protected amino group; and E4) reacting compound 54* with monosaccharide 5* to obtain compound 55* ##STR00434##  wherein P.sup.1, P.sup.3, P.sup.4, P.sup.6-P.sup.14 and P.sup.25 represent protecting groups, LG.sup.3 represents a leaving group and N.sub.p represents a protected amino group; and E5) Performing removal of protecting group P.sup.13 of compound 55* to obtain compound 56* ##STR00435##  wherein P.sup.1, P.sup.3, P.sup.4, P.sup.6-P.sup.12, P.sup.14 and P.sup.25 represent protecting groups, and N.sub.p represents a protected amino group; and E6) Reacting compound 56* with the disaccharide 19* to obtain compound 57* ##STR00436##  wherein P.sup.1, P.sup.3, P.sup.4, P.sup.6-P.sup.12, P.sup.14 and P.sup.16-P.sup.25 represent protecting groups, LG.sup.6 represents a leaving group and N.sub.p represents a protected amino group; and E7) Converting the protected amino groups of compound 57* to the corresponding acetamido groups to obtain compound 58* ##STR00437##  wherein P.sup.1, P.sup.3, P.sup.4, P.sup.6-P.sup.12, P.sup.14, P.sup.16-P.sup.21 and P.sup.25 represent protecting groups; and E8) Performing removal of protecting group P.sup.25 of compound 58* to obtain compound 59* and reacting compound 59* with alcohol HO-L-C in presence of a phosphorylating agent to obtain compound 15* ##STR00438## wherein P.sup.1, P.sup.3, P.sup.4, P.sup.6-P.sup.12, P.sup.14, P.sup.16-P.sup.22 represent protecting groups, and E9) Optionally performing removal of protecting group P.sup.21 of compound 15* to obtain compound 60* and reacting compound 60* with a phosphorylating agent to obtain compound 16* ##STR00439##  wherein P.sup.1, P.sup.3, P.sup.4, P.sup.6-P.sup.12, P.sup.14, P.sup.16-P.sup.20 and P.sup.22-P.sup.24 represent protecting groups, C represents -L-E.sub.p with E.sub.p being a solid support or a protected end group E; and E10) Performing removal of all remaining protecting groups from compound 15* or 16* to obtain compound 17* or 18* of general formula (I) ##STR00440## wherein L and E have the meanings as defined in claim 1.

17. An intermediate compound for preparing a saccharide of the general formula (I), wherein the intermediate compound has any one of general formulae (I2a), (I2b), (I2c), (I2d), (I2e), (I2f), (I4a), (I4b), (I4c), (I4d), (I4e), (I4f), (I4g), (I4h), (I4i), (I4j), (I5a), (I5b), (I5c), (I5d), (I5e), (I5f), (I5g), (I5h), (I5i), (I5j), (I6a), (I6b), (I6c), (I6d), (I6e), (I6f), (I6g), (I6h), (I7a), (I7b), (I7c), (I7d), (I7e), (I7f), (I7g), (I7h), (I7i), (I7j), (I7k), (I7m), (I7n), (I7o) or (I7p): ##STR00441## ##STR00442## ##STR00443## ##STR00444## ##STR00445## ##STR00446## ##STR00447## wherein P.sup.1-P.sup.25 represent protecting groups, Np represents a protected amino group, LG represents a leaving group and C represents -L-E.sub.p with Ep being a solid support or a protected end group E, wherein E and L have the meanings as defined in claim 1.

18. A method for raising a protective immune response in a human and/or animal host, the method comprising administering a saccharide according to claim 1, or a pharmaceutically acceptable salt thereof, to a subject in need thereof.

19. The method according to claim 18, wherein the saccharide is selected from the following: ##STR00448## wherein Z represents ##STR00449## or a pharmaceutically acceptable salt thereof.

20. The method according to claim 18, wherein the saccharide is a conjugate covalently linked to an immunogenic carrier through the residue E of the —O-L-E group, or a pharmaceutically acceptable salt thereof.

21. The method according to claim 20, wherein the conjugate has the following formula (V-2) ##STR00450## wherein L is —(CH.sub.2).sub.5—, E.sub.1 is —NH—, n is an integer selected from 1 or 2, c is comprised between 2 and 18, and —W— is: ##STR00451## and a represents an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10, or a pharmaceutically acceptable salt thereof.

22. A method for the prevention and/or treatment of diseases associated with bacteria containing in their cell-wall polysaccharide one of the following saccharide fragments: -6)-β-D-Glc-(1, 3)-β-D-GalNAc-(1, 4)-α-D-Glc-(1, 4)-[β-D-Glc-(1, 3)]-β-D-GalNAc-(1, 3)-α-D-Man-(1-; -3)-α-D-Man-(1, 6)-β-D-Glc-(1, 3)-β-D-GalNAc-(1, 4)-α-D-Glc-(1, 4)-[β-D-Glc-(1, 3)]-β-D-GalNAc-(1; -4)-[β-D-Glc-(1, 3)]-β-D-GalNAc-(1, 3)-α-D-Man-(1, 6)-β-D-Glc-(1, 3)-β-D-GalNAc-(1, 4)-α-D-Glc-(1; -4)-α-D-Glc-(1, 4)-[β-D-Glc-(1, 3)]-β-D-GalNAc-(1, 3)-α-D-Man-(1, 6)-β-D-Glc-(1, 3)-β-D-GalNAc-(1; and -3)-β-D-GalNAc-(1, 4)-α-D-Glc-(1, 4)-[β-D-Glc-(1, 3)]-β-D-GalNAc-(1, 3)-α-D-Man-(1, 6)-β-D-Glc-(1, the method comprising administering a saccharide according to claim 1, or a pharmaceutically acceptable salt thereof, to a subject in need thereof.

23. The method according to claim 22, wherein the saccharide is selected from the following: ##STR00452## wherein Z represents ##STR00453## or a pharmaceutically acceptable salt thereof.

24. The method according to claim 22, wherein the saccharide is a conjugate covalently linked to an immunogenic carrier through the residue E of the —O-L-E group; or a pharmaceutically acceptable salt thereof.

25. The method according to claim 24, wherein the conjugate has the following formula (V-2) ##STR00454## wherein L is —(CH.sub.2).sub.5—, E.sub.1 is —NH—, n is an integer selected from 1 or 2, c is comprised between 2 and 18, and —W— is: ##STR00455## and a represents an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10, or a pharmaceutically acceptable salt thereof.

26. The method according to claim 22, wherein the bacterium is Clostridium difficile.

27. A pharmaceutical composition comprising the saccharide according to claim 1, or a pharmaceutically acceptable salt thereof, together with at least one pharmaceutically acceptable adjuvant and/or excipient.

28. The pharmaceutical composition according to claim 27, wherein the saccharide is selected from the following: ##STR00456## wherein Z represents ##STR00457## or a pharmaceutically acceptable salt thereof.

29. The pharmaceutical composition according to claim 27, wherein the saccharide is a conjugate covalently linked to an immunogenic carrier through the residue E of the —O-L-E group; or a pharmaceutically acceptable salt thereof.

30. The pharmaceutical composition according to claim 29, wherein the conjugate has the following formula (V-2) ##STR00458## wherein L is —(CH.sub.2).sub.5—, E.sub.1 is —NH—, n is an integer selected from 1 or 2, c is comprised between 2 and 18, and —W— is: ##STR00459## and a represents an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10, or a pharmaceutically acceptable salt thereof.

Description

DESCRIPTION OF THE FIGURES

[0596] FIG. 1 shows the chemical structure of the repeating unit of C. difficile PS-II cell-wall saccharide.

[0597] FIG. 2 provides examples of functional group X of the interconnecting molecule according to the present invention.

[0598] FIG. 3 provides examples of functional group X of the interconnecting molecule according to the present invention.

[0599] FIG. 4 shows a CRM.sub.197 conjugate of the general formula (V-2) as preferred compounds of the present application.

[0600] FIG. 5 shows two paths how the compound 33 could be cleaved by NaOH treatment. Path I shows the cleavage at the phosphate group where the phosphate group remains at the linker part and compound LA, 5-aminopentyl dihydrogen phosphate, is formed. Path II shows the cleavage at the phosphate group where the phosphate group remains at the saccharide moiety (compound 33B) and compound LB, 5-aminopentane-1-ol, is formed.

[0601] FIG. 6 shows HPLC plots from bottom to top of the following compounds: Compound 33 (standard), compound 33A (control), compound 33 after one day treatment with 0.1 M sodium hydroxide solution at room temperature and purified, compound 33 after four days treatment with 0.1 M sodium hydroxide solution at room temperature, purified compound 33B, compound LB. It is evident from FIG. 7 that compound 33 is fully stable under basic conditions for one day. After four days of treatment with NaOH at rt still 50% of compound 33 remains intact.

[0602] FIG. 7 shows HPLC plots from bottom to top of the following compounds: compound 33A (control), compound 33 after one day treatment with 0.1 M sodium hydroxide solution at room temperature and purified, compound 33 (standard), compound 33 after two months at 2° C.-8° C. in water, compound 33 after two months at 2° C.-8° C. in NaPi which is a synonym for PBS (phosphate-buffered saline), compound 33 after two months at 2° C.-8° C. in Alhydrogel and PBS. It is evident from FIG. 7 that compound 33 is fully stable at 2° C. to 8° C. over two months.

[0603] FIG. 8 shows HPLC plots from bottom to top of the following compounds: compound 33A (control), compound 33 after one day treatment with 0.1 M sodium hydroxide solution at room temperature and purified, compound 33 (standard), compound 33 after two months at 25° C. in water, compound 33 after two months at 25° C. in NaPi which is a synonym for PBS (phosphate-buffered saline), compound 33 after two months at 25° C. in Alhydrogel and PBS. It is evident from FIG. 8 that compound 33 is fully stable at 25° C. over two months.

[0604] FIG. 9 shows HPLC plots from bottom to top of the following compounds: compound 33A (control), compound 33 after one day treatment with 0.1 M sodium hydroxide solution at room temperature and purified, compound 33 (standard), compound 33 after two months at 37° C. in water, compound 33 after two months at 37° C. in NaPi which is a synonym for PBS (phosphate-buffered saline), compound 33 after two months at 37° C. in Alhydrogel and PBS. It is evident from FIG. 9 that compound 33 is fully stable at 37° C. over two months.

[0605] FIG. 10 shows HPLC plots from bottom to top of the following compounds: compound 33A (control), compound 33 after one day treatment with 0.1 M sodium hydroxide solution at room temperature and purified, compound 33 (control), compound 92 (standard), compound 92 after one week at 2-8° C. in water, compound 92 after one week at 2-8° C. in NaPi which is a synonym for PBS (phosphate-buffered saline), compound 92 after one week at 2-8° C. in Alhydrogel and PBS. It is evident from FIG. 10 that compound 92 is fully stable at 2-8° C. over one week.

[0606] FIG. 11 shows HPLC plots from bottom to top of the following compounds: compound 33A (control), compound 33 after one day treatment with 0.1 M sodium hydroxide solution at room temperature and purified, compound 33 (control), compound 92 (standard), compound 92 after one week at 25° C. in water, compound 92 after one week at 25° C. in NaPi which is a synonym for PBS (phosphate-buffered saline), compound 92 after one week at 25° C. in Alhydrogel and PBS. It is evident from FIG. 11 that compound 92 is fully stable at 25° C. over one week.

[0607] FIG. 12 shows HPLC plots from bottom to top of the following compounds: compound 33A (control), compound 33 after one day treatment with 0.1 M sodium hydroxide solution at room temperature and purified, compound 33 (control), compound 92 (standard), compound 92 after one week at 37° C. in water, compound 92 after one week at 37° C. in NaPi which is a synonym for PBS (phosphate-buffered saline), compound 92 after one week at 37° C. in Alhydrogel and PBS. It is evident from FIG. 12 that compound 92 is fully stable at 37° C. over one week.

[0608] FIG. 13 shows HPLC plots from bottom to top of the following compounds: compound 33A (control), compound 33 after one day treatment with 0.1 M sodium hydroxide solution at room temperature and purified, compound 33 (control), compound 54 (standard), compound 54 after one week at 25° C. in water, compound 54 after one week at 2-8° C. in water, compound 54 after one week at 37° C. in water. It is evident from FIG. 13 that compound 54 is fully stable at 37° C. over one week.

[0609] FIG. 14 shows HPLC plots from bottom to top of the following compounds: compound 33A (control), compound 54 (standard), compound 54 after one week at 25° C. in Alhydrogel, compound 54 after one week at 2-8° C. in Alhydrogel, compound 54 after one week at 37° C. in Alhydrogel. It is evident from FIG. 14 that compound 54 when formulated with Alhydrogel becomes mostly adsorbed to the aluminum hydroxide and that no conceivable cleavage products were formed, which are detectable by HPLC in the presence of aluminium hydroxide. Thus compound 54 is stable at 37° C. over one week.

[0610] FIG. 15 shows ELISA titers of Day-0, Day-7 and Day-42 of pooled sera from rabbits (n=4) immunized with C. difficile saccharide 33-CRM.sub.197 formulations (36). The sera obtained from the rabbits immunized with compound 36 were diluted 1:100, 1000 with 1% BSA-PBS. The diluted sera (100 μL) were added per well of a microtiter plate which was coated with 0.5 μg of the corresponding 33-BSA conjugate (compound 37). Detection was done using HRP conjugated goat anti-rabbit secondary antibody diluted to 1:10000 and developed using 3,3′,5,5′-Tetramethylbenzidine (TMB) as a substrate. Absorbance was measured at 450 nm and the data were plotted using the Graphpad prism software. At day 42 a remarkable immunological response is evident from FIG. 15.

[0611] FIG. 16 shows ELISA titers of rabbit antisera against C. difficile strain 630 (pooled sera). Rabbits (4 animals per study arm) were immunized four times (days 0, 14, 28, 77) subcutaneously with 2.5 μg or 10 μg glycan antigen per injection with or without aluminum hydroxide (Alum) adjuvant, as indicated. PBS with Alum served as negative control. The immunogen was conjugate 56. Pooled sera from different timepoints (days 0, 7, 21, 35, 77 and 84) were tested for total IgG against formalin-inactivated C. difficile bacteria (strain 630) coated onto the ELISA plates. Coated ELISA plates purchased from tgcBIOMICS GmbH were blocked with 200 μL per well of commercial blocking reagent (Roche, ref. 11112589001) for 2 hours. Sera were diluted 1:100 with 1% (w/v) BSA in PBS and incubated for 1 hour at a volume of 100 μL per well. Total IgG was then detected using an HRP-conjugated goat anti-rabbit IgG secondary antibody (Sigma-Aldrich, ref. A4914) diluted to 1:10,000 in 1% (w/v) BSA in PBS for 30 min and developed using the TMB substrate (Thermo Scientific, ref. 34028). Absorbance was measured at 450 nm in a microplate reader and background-subtracted data were plotted using the GraphPad Prism software. It is evident from FIG. 16 that vaccination of rabbits with conjugate 56 induces IgG antibodies that bind to the surface of C. difficile bacteria, strain 630. Further, addition of Alum adjuvant leads to higher overall IgG titers.

[0612] FIG. 17 shows ELISA titers of rabbit antisera against C. difficile strain 630 (individual sera). Rabbits (4 animals per study arm) were immunized four times (days 0, 14, 28, 77) subcutaneously with 2.5 μg or 10 μg glycan antigen per injection with or without aluminum hydroxide (Alum) adjuvant, as indicated. PBS with Alum served as negative control. The immunogen was conjugate 56. Sera from different timepoints (days 0, 735, 77 and 84) were tested for total IgG against formalin-inactivated C. difficile bacteria (strain 630) coated onto the ELISA plates. Coated ELISA plates purchased from tgcBIOMICS GmbH were blocked with 200 μL per well of commercial blocking reagent (Roche, ref. 11112589001) for 2 hours. Sera were diluted 1:300 with 1% (w/v) BSA in PBS and incubated for 1 hour at a volume of 100 μL per well. Total IgG was then detected using an HRP-conjugated goat anti-rabbit IgG secondary antibody (Sigma-Aldrich, ref. A4914) diluted to 1:10,000 in 1% (w/v) BSA in PBS for 30 min and developed using the TMB substrate (Thermo Scientific, ref. 34028). Absorbance was measured at 450 nm in a microplate reader and background-subtracted data were plotted using the GraphPad Prism software. It is evident from FIG. 17 that vaccination of rabbits with conjugate 56 induces IgG antibodies that bind to the surface of C. difficile bacteria, strain 630. Further, addition of Alum adjuvant leads to higher overall IgG titers.

[0613] FIG. 18 shows ELISA titers of rabbit antisera against C. difficile strain R20291. Rabbits (4 animals per study arm) were immunized four times (days 0, 14, 28, 77) subcutaneously with 2.5 μg or 10 μg glycan antigen per injection with or without aluminum hydroxide (Alum) adjuvant, as indicated. PBS with Alum served as negative control. The immunogen was conjugate 56. Pooled sera from different timepoints (days 21, 35, 77 and 84) were tested for total IgG against formalin-inactivated C. difficile bacteria (strain R20291) coated onto the ELISA plates. Commercially available coated ELISA plates were blocked with 200 μL per well of commercial blocking reagent (Roche, ref. 11112589001) for 2 hours. Sera were diluted 1:100 with 1% (w/v) BSA in PBS and incubated for 1 hour at a volume of 100 μL per well. Total IgG was then detected using an HRP-conjugated goat anti-rabbit IgG secondary antibody (Sigma-Aldrich, ref. A4914) diluted to 1:10,000 in 1% (w/v) BSA in PBS for 30 min and developed using the TMB substrate (Thermo Scientific, ref. 34028). Absorbance was measured at 450 nm in a microplate reader and background-subtracted data were plotted using the GraphPad Prism software. It is evident from FIG. 18 that vaccination of rabbits with conjugate 56 induces IgG antibodies that bind to the surface of C. difficile bacteria, strain R20291. Further, addition of Alum adjuvant leads to higher overall IgG titers.

[0614] FIG. 19A shows ELISA titers of rabbit antisera (day 35) against C. difficile strain VPI10463. Rabbits (4 animals per study arm) were immunized four times (days 0, 14, 28, 77) subcutaneously with 2.5 μg or 10 μg glycan antigen per injection with or without aluminum hydroxide (Alum) adjuvant, as indicated. PBS with Alum served as negative control. The immunogen was conjugate 56. Pooled sera from day 35 were tested for total IgG against formalin-inactivated C. difficile bacteria (strain VPI10463) coated onto the ELISA plates. It is evident from FIG. 19A that vaccination of rabbits with conjugate 56 induces IgG antibodies that bind to the surface of C. difficile bacteria, strain VPI10463. Further, addition of Alum adjuvant leads to higher overall IgG titers.

[0615] FIG. 19B shows ELISA titers of rabbit antisera (day 35) against isolated C. difficile PS-II polysaccharide. Rabbits (4 animals per study arm) were immunized four times (days 0, 14, 28, 77) subcutaneously with 2.5 μg or 10 μg glycan antigen per injection with aluminum hydroxide (Alum) adjuvant, as indicated. PBS with Alum served as negative control. The immunogen was conjugate 56. Pooled sera from day 35 were tested for total IgG against isolated PS-II polysaccharide. It is evident from FIG. 19B that vaccination of rabbits with conjugate 56 induces IgG antibodies that bind to the isolated PS-II polysaccharide.

[0616] FIG. 19C shows ELISA titers of rabbit antisera (day 35) against C. difficile strain 630 with or without pre-incubation with isolated C. difficile PS-II polysaccharide. Rabbits (4 animals per study arm) were immunized four times (days 0, 14, 28, 77) subcutaneously with 10 μg glycan antigen per injection with aluminum hydroxide (Alum) adjuvant. The immunogen was conjugate 56. Pooled sera (diluted 1:100 in 1% (w/v) BSA in PBS) from day 35 were incubated on ice for 30 min with 10 or 50 μg of isolated PS-II polysaccharide or with PBS. The sera were then incubated for 1 hour (100 μL/well) on commercially available coated ELISA plates (C. difficile strain 630) that have been blocked beforehand with 200 μL per well of commercial blocking reagent (Roche, ref. 11112589001) for 2 hours. Total IgG was then detected using an HRP-conjugated goat anti-rabbit IgG secondary antibody (Sigma-Aldrich, ref. A4914) diluted to 1:10,000 in 1% (w/v) BSA in PBS for 30 min and developed using the TMB substrate (Thermo Scientific, ref. 34028). Absorbance was measured at 450 nm in a microplate reader and background-subtracted data were plotted using the GraphPad Prism software. It is evident from FIG. 19C that binding of rabbit antisera to C. difficile bacteria can be blocked with PS-II polysaccharide in a dose-dependent manner, indicating that anti-bacterial antibody responses are specific to the PS-II polysaccharide.

[0617] FIG. 20 shows ELISA titers of rabbit antisera against synthetic C. difficile PS-II hexasaccharide 54. Rabbits (4 animals per study arm) were immunized four times (days 0, 14, 28, 77) subcutaneously with 2.5 μg or 10 μg glycan antigen per injection with or without aluminum hydroxide (Alum) adjuvant, as indicated. PBS with Alum served as negative control. The immunogen was conjugate 56. Pooled sera from different timepoints (days 0, 21, 35, 77 and 84) were tested for total IgG against synthetic C. difficile PS-II hexasaccharide 54. It is evident from FIG. 20 that vaccination of rabbits with conjugate 56 induces IgG antibodies that bind to the synthetic immunogen 54. Further, addition of Alum adjuvant leads to higher overall IgG titers

[0618] FIG. 21 shows ELISA titers of rabbit antisera against C. difficile strain 630. Mice (7 or 8 animals per study arm) were immunized two times (days 0, 14, 28) subcutaneously with either conjugate 94 or conjugate 56 at a dose of 0.5 or 2 μg glycan antigen per injection. PBS served as negative control and aluminum hydroxide (Alum) adjuvant was used for all immunizations. Pooled sera from days 21 and 35 were tested for total IgG against formalin-inactivated C. difficile bacteria (strain 630) coated onto the ELISA plates. It is evident from FIG. 21 that vaccination of mice with conjugate 94 or 56 induces IgG antibodies that bind to the surface of C. difficile bacteria, strain 630.

[0619] FIG. 22 shows ELISA titers of rabbit antisera against C. difficile strain R20291. Mice (7 or 8 animals per study arm) were immunized two times (days 0, 14, 28) subcutaneously with either conjugate 94 or 56 at a dose of 0.5 or 2 μg glycan antigen per injection. PBS served as negative control and aluminum hydroxide (Alum) adjuvant was used for all immunizations. Pooled sera from days 21 and 35 were tested for total IgG against formalin-inactivated C. difficile bacteria (strain R20291) coated onto the ELISA plates. It is evident from FIG. 22 that vaccination of mice with conjugate 94 or 56 induces IgG antibodies that bind to the surface of C. difficile bacteria, strain 630.

[0620] FIG. 23 shows ELISA titers of mouse antisera against synthetic C. difficile PS-II antigens. Mice (7 or 8 animals per study arm) were immunized two times (days 0, 14, 28) subcutaneously with either conjugate 94 or conjugate 56 at a dose of 0.5 or 2 μg glycan antigen per injection. PBS served as negative control and aluminum hydroxide (Alum) adjuvant was used for all immunizations. Pooled sera from days 21 and 35 were tested for total IgG against the respective synthetic C. difficile glycan antigen that was used for immunization. It is evident from FIG. 23 that vaccination of mice with conjugate 94 or 56 induces IgG antibodies that bind to the synthetic immunogens. Further, addition of Alum adjuvant leads to higher overall IgG titers.

[0621] FIG. 24 shows SEC chromatograms of two glycoconjugates 94 and 56 used for immunization experiments. Unconjugated CRM.sub.197 protein served as control.

[0622] FIG. 25 shows SDS-PAGE of C. difficile glycoconjugates 94 and 56 (2.5 μg per well) used for immunization experiments resolved using a 10% polyacrylamide gel. Unconjugated CRM.sub.197 protein served as control.

[0623] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those skilled in the art that the techniques disclosed in the examples, which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those skilled in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments, which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

[0624] Further modifications and alternative embodiments of various aspects of the invention will be apparent to those skilled in the art in view of this description. Accordingly, this description is to be construed as illustrative only and is for the purpose of teaching those skilled in the art the general manner of carrying out the invention. It is to be understood that the forms of the invention shown and described herein are to be taken as examples of embodiments. Elements and materials may be substituted for those illustrated and described herein, parts and processes may be reversed, and certain features of the invention may be utilized independently, all as would be apparent to one skilled in the art after having the benefit of this description of the invention. Changes may be made in the elements described herein without departing from the spirit and scope of the invention as described in the following claims.

Examples

A. Chemical Synthesis

General Information:

[0625] Commercial grade solvents were used unless stated otherwise. Dry solvents were obtained from a Waters Dry Solvent System. Solvents for chromatography were distilled prior to use. Sensitive reactions were carried out in heat-dried glassware and under an argon atmosphere. Analytical thin layer chromatography (TLC) was performed on Kieselgel 60 F254 glass plates precoated with a 0.25 mm thickness of silica gel. Spots were visualized by staining with vanillin solution (6% (w/v) vanillin and 10% (v/v) sulfuric acid in 95% EtOH) or Hanessian's stain (5% (w/v) ammonium molybdate, 1% (w/v) cerium(II) sulfate and 10% (v/v) sulfuric acid in water). Silica column chromatography was performed on Fluka Kieselgel 60 (230-400 mesh).

[0626] .sup.1H, .sup.13C and two-dimensional NMR spectra were measured with a Varian 400-MR spectrometer at 296 K. Chemical shifts (d) are reported in parts per million (ppm) relative to the respective residual solvent peaks (CDCl.sub.3: d 7.27 in .sup.1H and 77.23 in .sup.13C NMR; CD.sub.3OD: d 3.31 in .sup.1H and 49.15 in .sup.13C NMR). The following abbreviations are used to indicate peak multiplicities: s singlet; d doublet; dd doublet of doublets; t triplet; dt doublet of triplets; q quartet; m multiplet. Coupling constants (J) are reported in Hertz (Hz). Optical rotation (OR) measurements were carried out with a Schmidt & Haensch UniPol L1000 polarimeter at λ=589 nm and a concentration (c) expressed in g/100 mL in the solvent noted in parentheses. High resolution mass spectrometry (HRMS) was performed at the Free University Berlin, Mass Spectrometry Core Facility, with an Agilent 6210 ESI-TOF mass spectrometer. Infrared (IR) spectra were measured with a Perkin Elmer 100 FTIR spectrometer.

A.1 Abbreviations

[0627] ACN acetonitrile [0628] AcOH acetic acid [0629] AIBN azobisisobutyronitrile [0630] Alhydrogel Aluminium Hydroxide Gel Adjuvant, Al: 10 mg/mL (Brenntag) [0631] Alloc allyloxycarbonyl [0632] aq. aqueous [0633] BH.sub.3 borane [0634] BBr.sub.3 boron tribromide [0635] Boc tert-butoxycarbonyl [0636] BnBr benzyl bromide [0637] br. broad [0638] CAS CAS Registry Number (CAS=Chemical Abstracts Service) [0639] CHCl.sub.3 chloroform [0640] cHex cyclohexane [0641] d doublet [0642] dd doublet of doublets [0643] DCM dichloromethane [0644] DDQ 2,3-dichloro-5,6-dicyano-1,4-benzoquinone [0645] DEAD diethyl azodicarboxylate [0646] DIPEA N,N-diisopropyl-ethylamine [0647] DMAP dimethylaminopyridine [0648] DME dimethoxyethane [0649] DMF dimethylformamide [0650] DMSO dimethylsulfoxide [0651] DPPA diphenylphosphoryl azide [0652] EDC.HCl N1-((ethylimino)methylene)-N3,N3-dimethylpropane-1,3-diamine hydrochloride [0653] ES electrospray [0654] Et.sub.2O diethyl ether [0655] EtOAc ethyl acetate [0656] FCS fetal calf serum [0657] FmocCl 9-fluorenylmethoxycarbonyl chloride [0658] GSDMD Gasdermin-D [0659] h hour [0660] HCl hydrochloric acid [0661] HEK293T embryonic kidney fibroblast cell line [0662] H.sub.2O water [0663] HOBt.H.sub.2O 1H-benzo[d][1,2,3]triazol-1-ol hydrate [0664] hPBMC human Peripheral Blood Mononuclear Cells [0665] IC.sub.50 half maximal inhibitory concentration [0666] K.sub.2CO.sub.3 potassium carbonate [0667] LDH lactate dehydrogenase [0668] LiAlH.sub.4 lithium aluminium hydride [0669] m multiplet [0670] MeCN acetonitrile [0671] MeOH methanol [0672] MeI methyl iodide [0673] MgSO.sub.4 magnesium sulphate [0674] min minutes [0675] MS mass spectrometry [0676] Na.sub.2CO.sub.3 sodium carbonate [0677] NaCNBH.sub.3 sodium cyanoborohydride [0678] NaHCO.sub.3 sodium hydrogencarbonate [0679] NaH sodium hydride [0680] NaOH sodium hydroxide [0681] NAP 2-naphthylmethyl [0682] NapBr 2-naphthylmethylbromide [0683] NaPi buffer phosphate-buffered saline (PBS) [0684] Na.sub.2SO.sub.4 sodium sulphate [0685] NBS N-bromosuccinimide [0686] NCS N-chlorosuccinimide [0687] NET neutrophil extracellular traps [0688] NIS N-iodosuccinimide [0689] NMR nuclear magnetic resonance [0690] PBBBr p-bromobenzylbromide [0691] PBS=NaPi phosphate-buffered saline [0692] Pd/C palladium on carbon [0693] Pd(PPh.sub.3).sub.4 Tetrakis(triphenylphosphine)palladium(0) [0694] PMA phorbol 12-myristate 13-acetate [0695] PPh.sub.3 triphenylphosphine [0696] PTFE polytetrafluoroethylene [0697] q quartet [0698] RBF round bottom flask [0699] rt room temperature [0700] s singlet [0701] sat. saturated [0702] sep septet [0703] t triplet [0704] TBAF tetrabutylammonium fluoride [0705] TFA trifluoroacetic acid [0706] THF tetrahydrofuran [0707] THP1 acute monocytic leukaemia cancer cell line [0708] TLC thin layer chromatography [0709] TMSOTf trimethylsilyl trifluoromethanesulfonate [0710] TsOH tosic acid [0711] Wt weight

A.2 Synthesis of Hexasaccharide 33

Synthesis of 2

[0712] ##STR00196##

[0713] NIS (3.0 equiv.) was added to a cooled solution of 1 (obtained according to Chem. Eur. J. 2014, 20, 3578-3583) in THF:H.sub.2O (4:1, 25 mL/1 g) at 0° C. After 10 min, reaction mixture was brought to rt and stirred for 2h. After complete consumption of starting material, THF was removed under reduced pressure and the obtained crude residue was dissolved in EtOAc and washed with aq. Na.sub.2S.sub.2O.sub.3 and aq. NaHCO.sub.3. Separated organic layer was dried over Na.sub.2SO.sub.4, concentrated and the crude product was purified by automated flash column chromatography on silica gel (0-60% EtOAc in cyclohexane) to afford the desired hemiacetal 2 (84%) as foam. HRMS (ESI+) Calculated for C.sub.38H.sub.38O.sub.6Na.sup.+ [M+Na].sup.+ 613.2566. found 613.2574.

Synthesis of 3

[0714] ##STR00197##

[0715] Ac.sub.2O (2.0 equiv.) and trimethylamine (6.0 equiv.) were added to a clear solution of 2 in DCM (10 mL/1 g) and kept for stirring at rt for 4h. After complete consumption of starting material, solvents were removed under vacuum and the crude product was purified by automated flash column chromatography on silica gel (0-50% EtOAc in cyclohexane) to afford the desired product 3 (94%) as viscous liquid. HRMS (ESI+) Calculated for C.sub.40H.sub.40O.sub.7Na.sup.+ [M+Na].sup.+ 655.2672. found 655.2679.

Synthesis of 4

[0716] ##STR00198##

[0717] Allyl trimethylsilane (2.0 equiv.) was added to a clear solution of 3 in dry acetonitrile (20 mL/1 g) at room temperature and followed by dropwise addition of TMSOTf (0.5 equiv.). The flask was sealed and placed in an ultrasonic cleaning bath (frequency 80 Hz, 100% power 230 V, rt) until the reaction was complete by TLC (40 min)). After complete consumption of starting material, the reaction mixture was quenched with aq. NaHCO.sub.3, diluted with EtOAc and washed with brine. The separated organic layers were dried over Na.sub.2SO.sub.4, concentrated and the crude product was purified by automated flash column chromatography on silica gel (0-60% EtOAc in cyclohexane) to afford the desired C-glycoside 4 as oil (91%). HRMS (ESI+) Calculated for C.sub.41H.sub.42O.sub.5Na.sup.+ [M+Na].sup.+ 637.2930. found 637.2929.

Synthesis of 5

[0718] ##STR00199##

[0719] PdCl.sub.2 (0.1 equiv.) was added to a degassed (30 min) solution of 4 in toluene (100 mL/1 g). After addition of PdCl.sub.2 the reaction mixture was degassed again for 30 min and kept for stirring at 120° C. for 2.5 d. After complete consumption of starting material, the reaction mixture was passed through celite pad and concentrated under reduced pressure. The crude residue was purified by automated flash column chromatography on silica gel (0-50% EtOAc in cyclohexane) to afford the double bond migrated compound 5 (70%) as yellowish liquid. HRMS (ESI+) Calculated for C.sub.41H.sub.42O.sub.5Na.sup.+ [M+Na].sup.+ 637.2930. found 637.2942.

Synthesis of 6

[0720] ##STR00200##

[0721] DDQ (1.2 equiv.) was added to a biphasic solution of 5 in DCM:H.sub.2O (19:1, 20 mL/1 g) at 0° C. After 10 min at 0° C., the reaction mixture was warmed to room temperature and stirred at room temperature for 1 h. After complete consumption of starting material, reaction mixture was diluted with DCM and extracted with aq. NaHCO.sub.3 and brine. The organic layer was dried over Na.sub.2SO.sub.4, filtered, and the filtrate was concentrated to obtain the crude product. The crude product was purified by automated flash chromatography on silica gel (0-80% EtOAc in cyclohexane) to give the desired product 6 as white oil (94%). HRMS (ESI+) Calculated for C.sub.30H.sub.34O.sub.5Na.sup.+ [M+Na].sup.+ 497.2304. found 497.2312.

Synthesis of 7

[0722] ##STR00201##

[0723] Ac.sub.2O (2.0 equiv.) and trimethylamine (6.0 equiv.) were added to a clear solution of 6 in DCM (10 mL/1 g) and kept for stirring at rt for 4 h. After complete consumption of starting material, solvents were removed under vacuum and the crude product was purified by automated flash column chromatography on silica gel (0-50% EtOAc in cyclohexane) to afford the desired product 7 (90%) as viscous liquid. HRMS (ESI+) Calculated for C.sub.32H.sub.36O.sub.6Na.sup.+ [M+Na].sup.+ 539.2410. found 539.2419.

Synthesis of 8

[0724] ##STR00202##

[0725] Ozone was bubbled through a cooled solution of 7 in DCM:MeOH (1:1, 170 mL/1 g) at −78° C. until a blue color was persisted. To remove residual O.sub.3, pure O.sub.2 was bubbled through the reaction mixture until the solution turned clear. Then, NaBH.sub.4 was added at −78° C., and the reaction mixture was stirred for 30 min at the same temperature. After complete consumption of starting material, the reaction mixture was quenched with aq. NH.sub.4Cl at −78° C. and washed with DCM. Separated organic layers were dried over Na.sub.2SO.sub.4 and concentrated under reduced pressure. The crude residue was purified by automated flash column chromatography on silica gel (0-100% EtOAc in cyclohexane) to afford the desired compound 8 (60% over 2 steps) as yellowish liquid. HRMS (ESI+) Calculated for C.sub.30H.sub.34O.sub.7Na.sup.+ [M+Na].sup.+ 529.2202. found 529.2220.

Synthesis of 9

[0726] ##STR00203##

[0727] To a solution of 8 in methanol (10 mL/1 g) was added sodium methoxide in MeOH (0.5 M, 10 mL) and the mixture was kept for stirring at rt for 1 h. After complete consumption of 8, AcOH (1 mL) was added until the pH of the reaction mixture was acidic. After neutralization, reaction mixture was concentrated, and the crude residue was purified by flash column chromatography (0-100%, EtOAc in cyclohexane) to give the desired compound 9 (90%) as paste. HRMS (ESI+) Calculated for C.sub.28H.sub.32O.sub.6Na.sup.+ [M+Na].sup.+ 487.2097. found 487.2111.

Alternative Synthesis of 9—Compound 10

[0728] ##STR00204##

[0729] Propargyltrimethylsilane (9.11 mL, 61.5 mmol, 2.0 equiv.) was added to a clear solution of 3 (19.5 g, 30.8 mmol) in dry acetonitrile (390 mL) at room temperature and followed by dropwise addition of TMSOTf (2.8 mL, 15.4 mmol, 0.5 equiv.). The flask was sealed and placed in an ultrasonic cleaning bath (frequency 80 Hz, 100% power 230 V, 5-10° C.) until the reaction was complete by TLC (40 min)). After complete consumption of starting material, the reaction mixture was quenched with aq. NaHCO3, diluted with EtOAc and washed with brine. The separated organic layers were dried over Na.sub.2SO.sub.4, concentrated and the crude product was purified by automated flash column chromatography on silica gel (0-60% EtOAc in cyclohexane) to afford the desired C-glycoside 10 as oil (16.2 g, 86%). HRMS (ESI+) Calcd for C.sub.41H.sub.40O.sub.5Na.sup.+ [M+Na].sup.+ 635.2773. found 635.2786.

Alternative Synthesis of 9—Compound 11

[0730] ##STR00205##

[0731] DDQ (18.7 g, 82.0 mmol, 1.2 equiv.) was added to a biphasic solution of 10 (42 g, 68.5 mmol) in DCM:H.sub.2O (19:1, 950 mL) at 0° C. After 10 min at 0° C., the reaction mixture was warmed to room temperature and stirred at room temperature for 1 h. After complete consumption of starting material, reaction mixture was diluted with DCM and extracted with aq. NaHCO.sub.3 and brine. The organic layer was dried over Na.sub.2SO.sub.4, filtered, and the filtrate was concentrated to obtain the crude product. The crude product was purified by automated flash chromatography on silica gel (0-80% EtOAc in cyclohexane) to give the desired product 11 as white oil (24 g, 74%, only α-isomer). HRMS (ESI+) Calcd for C.sub.30H.sub.32O.sub.5Na.sup.+ [M+Na].sup.+ 495.2147. found 495.2151.

Alternative Synthesis of 9—Compound 9

[0732] ##STR00206##

[0733] Ozone was bubbled through a cooled solution of 11 (10.6 g, 22.4 mmol) in DCM:MeOH (1:1, 1 L) at −78° C. until a blue color was persisted. To remove residual O.sub.3, pure O.sub.2 was bubbled through the reaction mixture until the solution turned clear. Then, NaBH.sub.4 (5.1 g, 135.0 mmol, 6.0 equiv.) was added at −78° C., and the reaction mixture was gradually brought to RT over 3 h and stirred at RT for 45 min. After complete consumption of starting material, the reaction mixture was quenched with aq. NH.sub.4Cl and washed with DCM three times. Separated organic layers were dried over Na.sub.2SO.sub.4 and concentrated under reduced pressure. The crude residue was purified by automated flash column chromatography on silica gel (0-100% EtOAc in cyclohexane) to afford the desired compound 9 (8.4 g, 81% over 2 steps) as oil (sticky white solid after drying under vacuum). HRMS (ESI+) Calcd for C.sub.28H.sub.32O.sub.6Na.sup.+ [M+Na].sup.+ 487.2097. found 487.2106.

Synthesis of 12

[0734] ##STR00207##

[0735] Sodium hydride (2.0 equiv., 60% in mineral oil) was added at 0° C. to a stirred solution of 9 in THF (20 mL/1 g). After 10 min, NapBr (1.05 equvi.) was added and the mixture was stirred for 24 h at 0° C. After 24 h, reaction mixture was quenched with MeOH, water, and extracted with EtOAc. The combined organic layers were washed with brine, dried over Na.sub.2SO.sub.4, filtered, and concentrated. The crude residue obtained was purified by automated flash column chromatography on silica gel (0-100%, EtOAc in cyclohexane) to give the desired compound 12 (54%) as paste. HRMS (ESI+) Calculated for C.sub.39H.sub.40O.sub.6Na.sup.+ [M+Na].sup.+ 627.2723. found 627.2748.

Synthesis of 14

[0736] ##STR00208##

[0737] Et.sub.3SiH (3.0 equiv.), TfOH (3.3 equiv.) were added to a cooled solution of 13 (obtained according to Org. Lett. 2011, 13, 378-381) in DCM (10 mL/1 g) with freshly activated molecular sieves (4 Å) at −78° C. The reaction mixture was stirred at the same temperature for 4 h. After complete consumption of starting material, reaction mixture was quenched with Et.sub.3N (1 mL) and diluted with DCM. The solution was washed with aq. NaHCO.sub.3 and brine. The organic layer was dried over Na.sub.2SO.sub.4, filtered and concentrated. The crude residue was purified by automated flash column chromatography on silica gel (0-100%, EtOAc in cyclohexane) to give the desired 4-OH compound 14 (83%) as white solid. HRMS (ESI+) Calculated for C.sub.51H.sub.54Cl.sub.3NO.sub.12NaS.sup.+ [M+Na].sup.+ 1034.2300. found 1034.2406.

Synthesis of 15

[0738] ##STR00209##

[0739] FmocCl (2.0 equiv.) and pyridine (3.0 equiv.) were added to a clear solution of 14 in DCM (10 mL/1 g) and kept for stirring at rt for 3.5 h. After complete consumption of starting material, reaction mixture was diluted with DCM and it was washed with brine. The organic layer was dried over Na.sub.2SO.sub.4, filtered and concentrated. The crude residue was purified by automated flash column chromatography on silica gel (0-100%, EtOAc in cyclohexane) to give the desired compound 15 (93%) as white solid. HRMS (ESI+) Calculated for C.sub.66H.sub.64Cl.sub.3NO.sub.14NaS.sup.+ [M+Na].sup.+ 1256.2981. found 1256.3125.

Synthesis of 16

[0740] ##STR00210##

[0741] NIS (1.4 equiv.) and TfOH (0.26 equiv.) were added to a cooled solution of acceptor 15 (1.0 equiv.) and donor 12 (1.2 equiv.) in DCM (0.06 M) in presence of 4 Å MS at −30° C. After 1.5 h, starting material was completely consumed, then Et.sub.3N (1.4 equiv.) was added and kept for stirring at rt for 2 h. After 2 h, reaction mixture was diluted with DCM and MS were filtered. The organic layer was washed with aq. Na.sub.2S.sub.2O.sub.3 and the separated organic layer was dried over Na.sub.2SO.sub.4, filtered and concentrated. The crude residue was purified by automated flash column chromatography on silica gel (0-100%, EtOAc in cyclohexane) to give the desired trisaccharide acceptor 16 (58% over 2 steps) as white solid. HRMS (ESI+) Calculated for C.sub.84H.sub.88Cl.sub.3NO.sub.18Na.sup.+ [M+Na].sup.+ 1528.4935. found 1528.5037.

Synthesis of 18

[0742] ##STR00211##

[0743] NIS (1.5 equiv.) and TfOH (0.4 equiv.) were added to a cooled solution of acceptor 16 (1.0 equiv.) and donor 17 (obtained according to J. Org. Chem. 2016, 81, 162-184) (1.5 equiv.) in toluene:dioxane (4:1, 0.03 M) in presence of 4 Å MS at 0° C. After 2 min, reaction mixture was kept at rt and stirred for 30 min. After 30 min, reaction mixture was quenched with Et.sub.3N, diluted with DCM and MS were filtered. The organic layer was washed with aq. Na.sub.2S.sub.2O.sub.3 and the separated organic layer was dried over Na.sub.2SO.sub.4, filtered and concentrated. The crude residue was purified by automated flash column chromatography on silica gel (0-100%, EtOAc in cyclohexane) to give the desired tetrasaccharide 18 (76%) as white solid. HRMS (ESI+) Calculated for C.sub.111H.sub.114Cl.sub.3NO.sub.23Na.sup.+ [M+Na].sup.+ 1958.6745. found 1958.6871.

Synthesis of 19

[0744] ##STR00212##

[0745] Et.sub.3SiH (3.0 equiv.), TfOH (3.3 equiv.) were added to a cooled solution of 18 in DCM (10 mL/1 g) in presence of freshly activated molecular sieves (4 Å) at −78° C. The reaction mixture was stirred at the same temperature for 4 h. After complete consumption of starting material, reaction mixture was quenched with Et.sub.3N (1 mL) and diluted with DCM. The solution was washed with aq. NaHCO.sub.3 and brine. The organic layer was dried over Na.sub.2SO.sub.4, filtered and concentrated. The crude residue was purified by automated flash column chromatography on silica gel (0-100%, EtOAc in cyclohexane) to give the desired tetrasaccharide 19 (82%) as white solid. HRMS (ESI+) Calculated for C.sub.111H.sub.116Cl.sub.3NO.sub.23Na.sup.+ [M+Na].sup.+ 1960.6901. found 1960.7024.

Synthesis of 21

[0746] ##STR00213##

[0747] Sodium hydride (2.0 equiv., 60% in mineral oil) was added at 0° C. to a stirred solution of 20 (obtained according to Tetrahedron: Asymmetry, 2000, 11, 481-492) in DMF (10 mL/1 g). After 10 min, PBBBr (1.1 equvi.) was added and the mixture was brought to rt. After stirring at rt for 1 h, reaction mixture was quenched with NH.sub.4Cl and extracted with EtOAc. The combined organic layers were washed with brine, dried over Na.sub.2SO.sub.4, filtered, and concentrated. The crude residue obtained was purified by automated flash column chromatography on silica gel (0-100%, EtOAc in cyclohexane) to give the desired compound 21 (62%) as paste. HRMS (ESI+) Calculated for C.sub.36H.sub.35BrO.sub.7NaS.sup.+ [M+Na].sup.+ 713.1185. found 713.1225.

Synthesis of 22

[0748] ##STR00214##

[0749] NBS (1.1 equiv.) and TMSOTf (0.1 equiv.) was added to a cooled solution of 21 in DCM:H.sub.2O (20:1, 10 mL/1 g) at 0° C. After 10 min, reaction mixture was quenched with aq., NaHCO.sub.3 and diluted with DCM. The organic layer was washed with brine. Separated organic layer was dried over Na.sub.2SO.sub.4, concentrated and the crude product was purified by automated flash column chromatography on silica gel (0-60% EtOAc in cyclohexane) to afford the desired hemiacetal 22 (70%) as foam. HRMS (ESI+) Calculated for C.sub.34H.sub.31BrO.sub.8Na.sup.+ [M+Na].sup.+ 669.1100. found 669.1132.

Synthesis of 23

[0750] ##STR00215##

[0751] Cs.sub.2CO.sub.3 (3.0 equiv.), CF.sub.3C(NPh)Cl (3.0 equiv.) were added to a stirred solution of 22 in DCM (10 mL/1 g) at 0° C. After 10 min., the mixture was brought to rt and stirred for 1 h. After complete consumption of 22, reaction mixture was filtered, and the filtrate was concentrated. The obtained crude residue was purified by automated flash column chromatography on silica gel (0-60% EtOAc in cyclohexane) to afford the desired imidate donor 23 (87%) as foam.

Synthesis of 25

[0752] ##STR00216##

[0753] The thioglycoside acceptor 24 was synthesized according to Danieli, E.; Lay, L.; Proietti, D.; Berti, F.; Costantino, P.; Adamo, R. Org Lett. 2011, 13, 378-381. TMSOTf in DCM (0.1 M, 0.2 equiv.) was added to a mixture of thioglycoside acceptor 24 (1.0 equiv.) and freshly dried 4 Å MS in DCM at −78° C. After 2 min, a solution of the imidate 23 (1.2 equiv.) in DCM was added. After 1 h, the reaction mixture was quenched with Et.sub.3N, and then filtered through a pad of Celite. The filtrate was concentrated, and the crude residue was purified by automated flash column chromatography on silica gel (0-100% EtOAc in cyclohexane) to afford the desired disaccharide 25 (61%) as solid. HRMS (ESI+) Calculated for C.sub.56H.sub.51BrCl.sub.3NO.sub.13NaS.sup.+ [M+Na].sup.+ 1186.1207. found 1186.1314.

Synthesis of 26

[0754] ##STR00217##

[0755] Et.sub.3SiH (3.0 equiv.), TfOH (3.3 equiv.) were added to a cooled solution of 25 in DCM (10 mL/1 g) with freshly activated molecular sieves (4 Å) at −78° C. The reaction mixture was stirred at the same temperature for 4 h. After complete consumption of starting material, reaction mixture was quenched with Et.sub.3N (1 mL) and diluted with DCM. The solution was washed with aq. NaHCO.sub.3 and brine. The organic layer was dried over Na.sub.2SO.sub.4, filtered and concentrated. The crude residue was purified by automated flash column chromatography on silica gel (0-100%, EtOAc in cyclohexane) to give the desired 4-OH compound 26 (80%) as white solid. HRMS (ESI+) Calculated for C.sub.56H.sub.53Cl.sub.3NBrO.sub.13NaS.sup.+ [M+Na].sup.+ 1188.1364. found 1188.1436.

Synthesis of 27

[0756] ##STR00218##

[0757] AcCl (2.0 equiv.) and pyridine (3.0 equiv.) were added to a clear solution of 26 in DCM (10 mL/1 g) at 0° C. and kept for stirring at rt for 3.5 h. After complete consumption of starting material, reaction mixture was diluted with DCM and it was washed with brine. The organic layer was dried over Na.sub.2SO.sub.4, filtered and concentrated. The crude residue was purified by automated flash column chromatography on silica gel (0-100%, EtOAc in cyclohexane) to give the desired compound 27 (70%) as white solid. HRMS (ESI+) Calculated for C.sub.58H.sub.55Cl.sub.3NBrO.sub.14NaS.sup.+ [M+Na].sup.+ 1230.1469. found 1230.1563.

Synthesis of 28

[0758] ##STR00219##

[0759] NIS (1.8 equiv.) and TfOH (0.4 equiv.) were added to a cooled solution of acceptor 19 (1.0 equiv.) and donor 27 (1.8 equiv.) in DCM (0.025 M) in presence of 4 Å MS at −20° C. Then the reaction mixture was gradually warmed to 0° C. during 3 h. After 3 h, reaction mixture was quenched with Et.sub.3N, diluted with DCM and MS were filtered. The organic layer was washed with aq. Na.sub.2S.sub.2O.sub.3 and the separated organic layer was dried over Na.sub.2SO.sub.4, filtered and concentrated. The crude residue was purified by automated flash column chromatography on silica gel (0-100%, EtOAc in cyclohexane) to give the desired hexasaccharide 28 (65%) as white solid. HRMS (ESI+) Calculated for C.sub.163H.sub.165Cl.sub.6N.sub.2BrO.sub.37Na.sup.+ [M+Na].sup.+ 3060.8258. found 3060.8275.

Synthesis of 29

[0760] ##STR00220##

[0761] To a clear solution of 28 in EtOAc (2.0 mM) were added Zn (100 equiv.), and AcOH (100 equiv.) and the reaction mixture was kept for stirring at room temperature 3 h. After complete consumption of starting material, reaction mixture was filtered through celite pad and concentrated. The residue obtained after solvents removal was dissolved in EtOAc (2.0 mM), Et.sub.3N (0.5 mL) and Ac.sub.2O (0.5 mL) were added. After stirring at rt for 2.5 d, the reaction mixture was concentrated. The crude obtained after solvent removal was dissolved in THF and methanol. To this clear solution 0.5 M NaOMe (3 mL) was added and kept for reflux at 65° C. After 16 h, reaction mixture was neutralized with AcOH and solvents were removed. The crude residue was purified by automated flash column chromatography on silica gel (0-100%, EtOAc in cyclohexane) to give the desired hexasaccharide 29 (74% over 3 steps) as white solid. HRMS (ESI+) Calculated for C.sub.143H.sub.155N.sub.2BrO.sub.31Na.sup.+ [M+Na].sup.+ 2500.9708. found 2500.9739.

Synthesis of 30

[0762] ##STR00221##

[0763] Ac.sub.2O (8.0 equiv.) and trimethylamine (8.0 equiv.) were added to a clear solution of 29 in DCM (10 mL/1 g) and kept for stirring at rt for 16 h. After complete consumption of starting material, solvents were removed under vacuum and the crude product was purified by automated flash column chromatography on silica gel (0-100% EtOAc in cyclohexane) to afford the desired product 30 (83%) as viscous liquid. HRMS (ESI+) Calculated for C.sub.151H.sub.163N.sub.2BrO.sub.35Na.sup.+ [M+Na].sup.+ 2669.0131. found 2669.0407.

Synthesis of 31

[0764] ##STR00222##

[0765] DDQ (1.1 equiv.) was added to a cooled solution of 30 in DCM:H.sub.2O at 0° C. After stirring the reaction mixture at the same temperature for 4 h, reaction was diluted with DCM and extracted with NaHCO.sub.3 aq. sat. solution and brine. The organic layer was dried over Na.sub.2SO.sub.4, filtered, and the filtrate was concentrated to obtain the crude product. The crude product was purified by automated flash column chromatography on silica gel (0-100% EtOAc in cyclohexane) to afford the desired product 31 (60%) as viscous liquid. HRMS (ESI+) Calculated for C.sub.140H.sub.155N.sub.2BrO.sub.35Na.sup.+ [M+Na].sup.+ 2527.9559. found 2527.9731.

Synthesis of 32

[0766] ##STR00223## ##STR00224##

[0767] To a solution of 31 in DCM, were added bis(diisopropylamino)-benzyloxyphosphine (2.0 equiv.) and diisopropylammonium tetrazolide (1.5 equiv.) and the solution was stirred at rt for 1.5 h. Then, 5-azido pentanol (8.0 equiv.) and tetrazole (9.0 equiv. 0.45 M solution in CAN) were added and kept for stirring at room temperature for 2 h. After 2 h, t-butyl peroxide (6.0 equiv., 5.0-6.0 M solution in decane) was added and the reaction mixture stirred for 1 h. After 1 h, reaction mixture was diluted with DCM and quenched with NaHCO.sub.3 aq. sat. solution. The aqueous layer was extracted with DCM. The combined organic layer was washed with brine, dried over Na.sub.2SO.sub.4, filtered, and the filtrate was concentrated under vacuum to obtain the crude product. The crude product was purified by automated flash column chromatography on silica gel (0-100% EtOAc in cyclohexane) to afford the desired product 32 (37% over 3 steps) as viscous liquid. MALDI Calculated for C.sub.152H.sub.171N.sub.5BrO.sub.38PH.sup.+ [M+H].sup.+ 2786.0635. found 2786.870.

Synthesis of 33

[0768] ##STR00225## ##STR00226##

[0769] Pd/C (6 mg) was added to a clear solution of 32 (6 mg) in EtOAc:MeOH:H.sub.2O:AcOH. Obtained inhomogeneous mixture was stirred under hydrogen atmosphere at rt for 40 h. After 40 h, reaction mixture was filtered through PTFE filter and concentrated under vacuum at 30° C. bath temperature of rotary evaporator for 10 min to remove methanol, EtOAc, AcOH and water. The crude product obtained after solvents removal was dissolved in MeOH, water and to this LiOH (2 N in water) was added at 0° C. The reaction mixture was stirred at 0° C. for 3 h. After 3 h, the reaction mixture was quenched with AcOH (30 μL) and the solvents were removed under reduced pressure and the obtained crude residue was purified with C18 reverse phase column chromatography using water and acetonitrile as solvents to give the desired final compound 33 (80% over 2 steps) as a white solid. HRMS (ESI+) Calculated for C.sub.46H.sub.82N.sub.3PO.sub.34.sup.+ [M−Na+2H].sup.+ 1252.4551. found 1252.4578.

Synthesis of 34

[0770] ##STR00227## ##STR00228##

[0771] Pd/C (2 mg) was added to a clear solution of 29 in EtOAc:MeOH:H.sub.2O:AcOH and the obtained inhomogeneous mixture was stirred under hydrogen atmosphere at rt for 40 h. After 40 h, reaction mixture was filtered through PTFE filter and concentrated under vacuum at 30° C. bath temperature of rotary evaporator for 10 min to remove methanol, EtOAc, AcOH and water. The crude product was purified with C18 reverse phase column chromatography using water and acetonitrile as solvents to give the desired final compound 34 (82%) as a white solid. HRMS (ESI+) Calculated for C.sub.41H.sub.70N.sub.2O.sub.31.sup.+ [M+Na].sup.+ 1109.3860. found 1109.3853.

Conjugation of 33 with CRM.sub.197 or BSA

##STR00229## ##STR00230##

[0772] Antigen 33 (1.0 equiv.) was dissolved in DMSO-H.sub.2O at rt in a 2 mL vial. Triethylamine (35.0 equiv.) was added to it. The mixture was added to the activated adipate-NHS ester (10 equiv.) in DMSO in an Eppendorf vial and stirred for 3 h at rt. The Antigen-NHS ester was precipitated out by adding 10 volume of EtOAc and centrifuged, supernatant was removed carefully. Washed the precipitate with EtOAc (1 mL×3), dried and taken for the next step. 1 mg of protein in NaPi buffer (˜100 μL) was added to reaction vial containing the Antigen-NHS ester 35 in 50 μL of NaPi buffer (pH 7.0) dropwise. The vial was finally rinsed with 50 μL of buffer solution and transferred to the reaction vial completely. The reaction mixture was stirred at rt for 22 h. Antigen-protein conjugate solution was transferred to the Amicon Ultra-0.5 mL, centrifuged for 6 minutes at room temperature. Added 300 μL of buffer to the reaction vial, rinsed and transferred to the filter and centrifuged again. Additional washings were done using 1×PBS solution for three more times. After the final wash the conjugate was stored in 1×PBS solution at 2-8° C. The conjugates were analysed using MALDI, (loading of 4-12 antigens on protein was obtained), SDS-page, BCA estimation, SEC-HPLC.

A.3 Synthesis of Hexasaccharide 54

Synthesis of 41

[0773] ##STR00231##

[0774] TBDPSCl (1.1 equiv.) and trimethylamine (2.8 equiv.) were added to a clear solution of 20 in CH.sub.3CN (10 mL/1 g) and kept for stirring at rt for 10 h. After complete consumption of starting material, solvents were removed under vacuum and the crude product was purified by automated flash column chromatography on silica gel (0-100% EtOAc in cyclohexane) to afford the desired product 41 (93%) as viscous liquid. HRMS (ESI+) Calculated for C.sub.45H.sub.48O.sub.7SSiNa.sup.+ [M+Na].sup.+ 783.2788. found 783.2767.

Synthesis of 42

[0775] ##STR00232##

[0776] The procedure described for the synthesis of compound 22 used for the synthesis of compound 42 (94%). HRMS (ESI+) Calculated for C.sub.43H.sub.44O.sub.8SiNa.sup.+ [M+Na].sup.+ 739.2703. found 739.2700.

Synthesis of 43

[0777] ##STR00233##

[0778] To a cooled solution of 42 in DCM at 0° C. was added trichloroacetonitrile (6.0 equiv.) and DBU (0.2 equiv.). After 3 h at 0° C., the reaction was complete, and the solvent was evaporated. The crude product was purified by automated flash column chromatography on silica gel (0-100% EtOAc in cyclohexane) to afford the desired product 43 (83%) as viscous liquid.

Synthesis of 44

[0779] ##STR00234##

[0780] The procedure described for the synthesis of compound 25 used for the synthesis of compound 44 (40%). HRMS (ESI+) Calculated for C.sub.65H.sub.64O.sub.13SiSNCl.sub.3Na.sup.+ [M+Na].sup.+ 1256.2801. found 1256.2645.

Synthesis of 45

[0781] ##STR00235##

[0782] The procedure described for the synthesis of compound 26 used for the synthesis of compound 45 (60%). HRMS (ESI+) Calculated for C.sub.65H.sub.66O.sub.13SiSNCl.sub.3Na.sup.+ [M+Na].sup.+ 1256.2987. found 1256.2974.

Synthesis of 46

[0783] ##STR00236##

[0784] The procedure described for the synthesis of compound 27 used for the synthesis of compound 46 (60%). HRMS (ESI+) Calculated for C.sub.67H.sub.68O.sub.14SiSNCl.sub.3Na.sup.+ [M+Na].sup.+ 1300.3064. found 1300.3090.

Synthesis of 47

[0785] ##STR00237##

[0786] The procedure described for the synthesis of compound 28 used for the synthesis of compound 47 (82%). HRMS (ESI+) Calculated for C.sub.172H.sub.178O.sub.37SiN.sub.2Cl.sub.6Na.sup.+ [M+Na].sup.+ 3127.9728. found 3127.9728.

Synthesis of 48

[0787] ##STR00238##

[0788] The procedure described for the synthesis of compound 29 used for the synthesis of compound 48 (50%). HRMS (ESI+) Calculated for C.sub.152H.sub.168O.sub.31SiN.sub.2Na.sup.+ [M+Na].sup.+ 2568.1298. found 2568.1322.

Synthesis of 49

[0789] ##STR00239## ##STR00240##

[0790] The procedure described for the synthesis of compound 30 used for the synthesis of compound 49 (80%). HRMS (ESI+) Calculated for C.sub.160H.sub.176O.sub.35SiN.sub.2Na.sup.+ [M+Na].sup.+ 2737.1754. found 2737.2001.

Synthesis of 50

[0791] ##STR00241##

[0792] The procedure described for the synthesis of compound 31 used for the synthesis of compound 50 (70%). HRMS (ESI+) Calcd for C.sub.149H.sub.168O.sub.35SiN.sub.2Na.sup.+ [M+Na].sup.+ 2596.1095. found 2595.9954 and 2596.9997.

Synthesis of 51

[0793] ##STR00242##

[0794] The procedure described for the synthesis of compound 32 used for the synthesis of compound 51.

Synthesis of 52

[0795] ##STR00243##

[0796] A premixed solution of TBAF and AcOH was added to a clear solution of 51 in THF at rt and the reaction mixture was kept for stirring at rt for 3 h. After complete consumption of starting material, reaction mixture was diluted with DCM and concentrated under vacuum to obtain the crude product. The crude product was purified by automated column chromatography on silica gel using EtOAc in n-hexane (gradient, 0 to 100%) as the eluent.

Synthesis of 53

[0797] ##STR00244##

[0798] To a solution of 52 in DCM, were added dibenzyl N,N-diisopropylphosphoramidite (2.0 equiv.) and diisopropylammonium tetrazolide (1.5 equiv.) and the solution was stirred at rt for 1.5 h. Then, t-butyl peroxide (6.0 equiv., 5.0-6.0 M solution in decane) was added and the reaction mixture stirred for 1 h. After 1 h, reaction mixture was diluted with DCM and quenched with NaHCO.sub.3 aq. sat. solution. The aqueous layer was extracted with DCM. The combined organic layer was washed with brine, dried over Na.sub.2SO.sub.4, filtered, and the filtrate was concentrated under vacuum to obtain the crude product. The crude product was purified by automated flash column chromatography on silica gel (0-100% EtOAc in cyclohexane) to afford the desired product 53.

Synthesis of 54

[0799] ##STR00245##

[0800] The procedure described for the synthesis of compound 33 used for the synthesis of compound 54.

Conjugation of 54 with CRM.sub.197 and BSA

##STR00246##

[0801] The procedure described for the synthesis of glycoconjugates 36 and 37 was also used for the synthesis of 56 and 57.

A.4 Alternative Synthesis of Hexasaccharide 54

Synthesis of 58

[0802] ##STR00247##

[0803] Diphenyl phosphite was added to a clear solution of 48 in pyridine, and the reaction mixture was stirred at room temperature under nitrogen for 2 h. After 2 h, 1 M TEAB solution was added to the reaction mixture at 0° C. After 5 min, ice bath was removed and the stirring was continued for another 2 h at rt. After complete consumption of starting material, reaction mixture was diluted with DCM and the organic layer was washed successively with 1 M TEAB solution and concentrated under reduced pressure. The crude product was purified by automated flash column chromatography (EA:DCM:MeOH with 2% Et.sub.3N) to give pure H-phosphonate derivative 58 (90%) as viscous liquid. HRMS (ESI+) Calcd for C.sub.155H.sub.184N.sub.3PSiO.sub.37.sup.+ [M].sup.+ 2740.2189. found 2740.2132.

Synthesis of 59

[0804] ##STR00248##

[0805] H-phosphonate 58 (1.0 equiv.) and linker (4.0 equiv.) were co-evaporated with pyridine and dried under vacuum for 30 min. After that, it was dissolved in py and to this PivCl (2.0 equiv.) was added. The reaction mixture was kept for stirring at rt for 2 h. After 2 h, the reaction was cooled to −40° C., a freshly prepared solution of I.sub.2 in Py:H.sub.2O (20:1) was added and the reaction mixture was kept for stirring at the same temperature for 1.5 h and later brought to rt and stirred at rt for 15 min. Then, TEAB (10 mL) was added to the mixture and diluted with dichloromethane, washed successively with 10% aq. sodium thiosulfate, 1 M aq. triethylammonium hydrogen carbonate (TEAB), dried over Na.sub.2SO.sub.4, filtered and concentrated. The residue was purified by automated flash column chromatography (ethyl acetate:DCM:MeOH) together with 2% trimethylamine as eluents give the desired product 59 (70%) as viscous liquid. Maldi (ESI+) Calcd for C.sub.154H.sub.178N.sub.5PNaSiO.sub.38.sup.+ [M-Et.sub.3N+Na].sup.+ 2789.1635. found 2788.0.

Synthesis of 60

[0806] ##STR00249##

[0807] To a solution of 59 in DCM and pyridine at 0° C. was added HF solution (70% in pyridine, 0.3 mL) drop wisely. The reaction mixture was stirred at the same temperature for 18 h. Then, the reaction mixture was diluted with DCM, washed with saturated aqueous NaHCO.sub.3 solution, and TEAB buffer. The organic phase was separated and dried (Na.sub.2SO.sub.4) and concentrated under reduced pressure. The residue was purified by automated flash column chromatography (ethyl acetate:DCM:MeOH) together with 2% trimethylamine as eluents give the desired product 60 as viscous liquid. Maldi (ESI+) Calcd for C.sub.138H.sub.160N.sub.5PNaO.sub.38.sup.+ [M-Et.sub.3N+Na].sup.+ 2550.7585. found 2549.698.

Synthesis of 54

[0808] ##STR00250##

[0809] To a solution of 60 in DCM was added dibenzyl diisopropylphosphoramidite (2.0 equiv.) and diisopropylammonium tetrazolide (2.0 equiv.) and the solution stirred at room temperature for 1.5 h. Then, t-butyl peroxide 5.0-6.0 M solution in decane (6.0 equiv.) was added at room temperature and the reaction mixture stirred for 1 h. The reaction mixture was diluted with DCM and washed with NaHCO.sub.3 aq. sat. solution and TEAB buffer. The aqueous layer was extracted with DCM (2×10 mL). The combined organic layer was dried over Na.sub.2SO.sub.4 (0.5 g), filtered, and the filtrate was concentrated under vacuum to obtain the crude product. The crude product was purified by automated flash chromatography using EtOAc:DCM:MeOH with 2% trimethylamine to obtain the desired product 61 as viscous oil (91%). Title compound 54 was obtained in 60% yield from compound 61 by the procedure described for the synthesis of compound 33. HRMS (ESI+) Calcd for C.sub.46H.sub.83N.sub.3P.sub.2NaO.sub.37.sup.+ [M+Na].sup.− 1354.4078. found 1354.9623.

A.5 Alternative Synthesis of Hexasaccharide 54

Synthesis of 62

[0810] ##STR00251##

[0811] Me.sub.3N.BH.sub.3 (21.2 g, 291 mmol, 5.4 equiv.), BF.sub.3.Et.sub.2O (42.2 mL, 291 mmol, 5.4 equiv.) were added to a cooled solution of 24 (28.8 g, 54 mmol) in CH.sub.3CN (1.5 L) at 0° C. The reaction mixture was stirred at the same temperature for 1 h. After complete consumption of starting material, reaction mixture was quenched with Et.sub.3N (30 mL) and MeOH (50 mL). Then Reaction mixture was diluted with EtOAc (1 L), washed with 1 M HCl (three times, sometimes it is difficult to see 2 layers then add brine to get better) and followed by aq. NaHCO3 until pH of the organic layer becomes neutral. The separated organic layer was dried over Na.sub.2SO.sub.4, filtered and concentrated. The product 62 (22 g, 76%) white solid was pure used for the next step. HRMS (ESI+) Calcd for C.sub.22H.sub.24Cl.sub.3NO.sub.6SNa.sup.+ [M+Na].sup.+ 558.0288. found 558.0332.

Synthesis of 64

[0812] ##STR00252##

[0813] The procedure described for the synthesis of compound 2 used for the synthesis of compound 64 (85%). HRMS (ESI+) Calcd for C.sub.34H.sub.40O.sub.6N.sup.+ [M+NH.sub.4].sup.+ 558.2856. found 558.2976.

Synthesis of 65

[0814] ##STR00253##

[0815] To a stirred solution of 64 (24.5 g, 45.3 mmol) in anhydrous DCM (360 mL), anhydrous DMF (1 mL, 13.6 mmol, 0.30 equiv.) and (COCl).sub.2 (10.3 mL, 118.0 mmol, 2.6 equiv.) were added at 0° C. After 5 min. reaction mixture was brought to rt and stirred at r.t. for 2 h. After complete consumption of starting material the reaction mixture was cooled to 0° C. quenched with Et.sub.3N. The salt formed was filtered through short pad of celite and washed with DCM (Do not wash with lot of DCM, salt will dissolve and pass through celite). Then, the filtrate was concentrated under reduced pressure and purified by silica gel column chromatography using ethyl acetate:cyclohexane (0-40% with 2% Et.sub.3N) to afford the desired glycosyl chloride 65 (24 g, 96%) as the viscous liquid. HRMS (ESI+) Calcd for C.sub.34H.sub.35O.sub.5ClNa.sup.+ [M+Na].sup.+581.2071. found 581.2206.

Synthesis of 66

[0816] ##STR00254##

[0817] To a turbid of glycosyl chloride 65 (16.2 g, 28.9 mmol, 1.15 equiv.) and acceptor 62 (13.5 g, 25.1 mmol) in acetonitrile (200 mL) and DCM (80 mL), were added Ag.sub.2O (8.8 g, 37.7 mmol, 1.5 equiv. dried under vacuum at 80° C. for 3 h before use) and 2-aminoethyl diphenylborinate (0.57 g, 2.51 mmol, 0.1 equiv.). After being stirred at rt. for 16 h, the mixture was diluted with DCM (80 mL), acetone (80 mL) and filtered through celite, sand and washed with DCM and Acetone till the filtrate showed no product. All the filtrate fractions were combined and concentrated. The residue was dissolved in EtOAc (300 mL) and kept at 55° C. till the solid dissolves and becomes the clear solution. Then this clear solution was filtered through filter paper and washed with hot EtOAc and kept for recrystallization. After 1 h white solid was crystalized and it was separated from the solution to give the desired disaccharide 66 as white solid (22 g, 83%). HRMS (ESI+) Calcd for C.sub.56H.sub.58Cl.sub.3NO.sub.11SNa.sup.+ [M+Na].sup.+ 1080.2696. found 1080.2904.

Synthesis of 67

[0818] ##STR00255##

[0819] FmocCl (16.87 g, 63.2 mmol, 2.0 equiv.) and pyridine (7.67 mL, 95.0 mmol, 3.0 equiv.) were added to a clear solution of 66 (33.5 g, 31.6 mmol) in DCM (330 mL) and kept for stirring at rt for 2 h. After complete consumption of starting material, reaction mixture was diluted with DCM and it was washed with brine. The organic layer was dried over Na.sub.2SO.sub.4, filtered and concentrated. The crude residue was purified by automated flash column chromatography on silica gel (0-100%, EtOAc in cyclohexane) to give the desired compound 67 (34.7 g, 86%) as white solid. HRMS (ESI+) Calcd for C.sub.71H.sub.68Cl.sub.3NO.sub.13SNa.sup.+ [M+Na].sup.+ 1302.3375. found 1302.3694.

Synthesis of 69

[0820] ##STR00256##

[0821] The procedure described for the synthesis of compound 2 used for the synthesis of compound 69 (80%). HRMS (ESI+) Calcd for C.sub.29H.sub.36O.sub.7N.sup.+ [M+NH.sub.4].sup.+ 510.2492. found 510.2527.

Synthesis of 70

[0822] ##STR00257##

[0823] To a stirred solution of 69 (18.0 g, 36.5 mmol) in anhydrous DCM (290 mL), anhydrous DMF (0.85 mL, 11.0 mmol, 0.30 equiv.) and (COCl).sub.2 (8.3 mL, 95.0 mmol, 2.6 equiv.) were added at 0° C. After 5 min. reaction mixture was brought to rt and stirred at r.t. for 2 h. After complete consumption of starting material the reaction mixture was cooled to 0° C. quenched with Et.sub.3N. The salt formed was filtered through short pad of celite and washed with DCM. Then, the filtrate was concentrated under reduced pressure and purified by silica gel column chromatography using ethyl acetate:cyclohexane (0-40% with 2% Et.sub.3N) to afford the desired glycosyl chloride 70 (16.7 g, 89%) as the viscous liquid. HRMS (ESI+) Calcd for C.sub.29H.sub.31O.sub.6ClNa.sup.+ [M+Na].sup.+ 533.1707. found 533.1752.

Synthesis of 71

[0824] ##STR00258##

[0825] To a turbid of glycosyl chloride 70 (16.5 g, 32.3 mmol, 1.15 equiv.) and acceptor 62 (15.07 g, 28.1 mmol) in acetonitrile (200 mL) and DCM (80 mL), were added Ag.sub.2O (9.76 g, 42.1 mmol, 1.5 equiv. dried under vacuum at 80° C. for 3 h before use) and 2-aminoethyl diphenylborinate (0.63 g, 2.81 mmol, 0.1 equiv.). After being stirred at rt. for 16 h, the mixture was diluted with DCM (80 mL), acetone (80 mL) and filtered through celite, sand and washed with DCM and Acetone till the filtrate showed no product. All the filtrate fractions were combined and concentrated. The residue was dissolved in EtOAc (400 mL) and kept at 55° C. till the solid dissolves and becomes the clear solution. Then this clear solution was filtered through filter paper and washed with hot EtOAc and kept for recrystallization. After 1 h white solid was crystalized and it was separated from the solution to give the desired disaccharide 71 as white solid (23 g, 81%). HRMS (ESI+) Calcd for C.sub.51H.sub.54Cl.sub.3NO.sub.12SNa.sup.+ [M+Na].sup.+ 1032.2330. found 1032.2423.

Synthesis of 72

[0826] ##STR00259##

[0827] AcCl (40 mL) was added to a turbid of 71 (18.87 g, 18.66 mmol) in MeOH (200 mL) and DCM (200 mL) at 0° C. After 5 minutes, ice bath was removed and kept at rt for stirring. After stirring at room temperature for 3 h, the reaction mixture was diluted with DCM and washed with water and aq. NaHCO.sub.3. The separated organic layer was dried over Na.sub.2SO.sub.4, filtered and concentrated on a rotary evaporator to yield the desired compound 72 (18.09 g, quantitative) as white solid. HRMS (ESI+) Calcd for C.sub.49H.sub.52Cl.sub.3NO.sub.11SNa.sup.+ [M+Na].sup.+ 990.2224. found 990.2301.

Synthesis of 73

[0828] ##STR00260##

[0829] To a suspension of 72 (18.05 g, 18.6 mmol) in acetonitrile (370 mL) was added imidazole (3.56 g, 52.3 mmol, 2.8 equiv.) and TBDPSCl (7.2 mL, 28.0 mmol, 1.5 equiv.). After 5 minutes reaction mixture was completely clear and left for stirring at rt for 30 minutes. After 30 minutes, the reaction mixture was diluted with EtOAc and washed with brine. The separated organic layer was dried over Na.sub.2SO.sub.4, filtered and concentrated. The crude residue obtained after solvents removal was purified by automated silica gel flash chromatography using ethyl acetate and cyclohexane as the eluents to give the desired product 73 (20.9 g, 93%) as solid. HRMS (ESI+) Calcd for C.sub.65H.sub.70Cl.sub.3NO.sub.11SSiNa.sup.+ [M+Na].sup.+ 1228.3402. found 1228.3481.

Synthesis of 74

[0830] ##STR00261##

[0831] To a clear solution of 73 (18.69 g, 15.47 mmol) in DCM (200 mL) were added Et.sub.3N (19 mL, 139.0 mmol, 9.0 equiv.), aceticanhydride (4.4 mL, 46.4 mmol, 3.0 equiv.) and DMAP (0.189 g, 1.547 mmol, 0.1 equiv.) and kept for stirring at rt for 18 h. After 18 h, reaction mixture was diluted with DCM and washed with aq. NaHCO.sub.3. The separated organic layer dried over Na.sub.2SO.sub.4 and concentrated. The crude residue obtained after solvents removal was purified by automated flash chromatography on silica gel (cyclohexane-EtOAc) to yield the desired product 74 as foam (17.2 g, 89%). HRMS (ESI+) Calcd for C.sub.67H.sub.72Cl.sub.3NO.sub.12SSiNa.sup.+ [M+Na].sup.+ 1272.3478. found 1272.3530.

Synthesis of 76

[0832] ##STR00262##

[0833] The procedure described for the synthesis of compound 16 used for the synthesis of compound 76 (60% over 2 steps). HRMS (ESI+) Calcd for C.sub.82H.sub.86O.sub.17NNaCl.sub.3.sup.+ [M+Na].sup.+ 1574.5329. found 1574.5624.

Synthesis of 77

[0834] ##STR00263##

[0835] The procedure described for the synthesis of compound 18 used for the synthesis of compound 77 (80%). HRMS (ESI+) Calcd for C.sub.116H.sub.122O.sub.22N2Cl.sub.3.sup.+ [M+NH.sub.4].sup.+ 2000.7565. found 2000.7588.

Synthesis of 78

[0836] ##STR00264##

[0837] The procedure described for the synthesis of compound 19 used for the synthesis of compound 78 (80%). HRMS (ESI+) Calcd for C.sub.116H.sub.124O.sub.22N.sub.2Cl.sub.3.sup.+ [M+NH.sub.4].sup.+ 2001.7711. found 2001.6469.

Synthesis of 79

[0838] ##STR00265##

[0839] The procedure described for the synthesis of compound 28 used for the synthesis of compound 79 (79%). HRMS (ESI+) Calcd for C.sub.177H.sub.186O.sub.34N.sub.2Cl.sub.6Na.sup.+ [M+Na].sup.+ 3147.0689. found 3147.1184.

Synthesis of 80

[0840] ##STR00266##

[0841] To a clear solution of 79 in EtOAc (2.0 mM) were added Zn (100 equiv.), AcOH (100 equiv.), Ac.sub.2O and the reaction mixture was kept for stirring at room temperature 20 h. After complete consumption of starting material, reaction mixture was filtered through celite pad and concentrated. The crude residue was purified by automated flash column chromatography on silica gel (0-100%, EtOAc in cyclohexane) to give the desired hexasaccharide 80 (69%) as white solid. HRMS (ESI+) Calcd for C.sub.175H.sub.188N.sub.2O.sub.32Si.sup.+ [M].sup.+ 2858.2948. found 2858.3062.

Synthesis of 81

[0842] ##STR00267##

[0843] The procedure described for the synthesis of compound 31 used for the synthesis of compound 81 (73%). HRMS (ESI+) Calcd for C.sub.164H.sub.180O.sub.32N.sub.2Si.sup.+ [M].sup.+ 2718.2322, found 2718.2347.

Synthesis of 82

[0844] ##STR00268##

[0845] The procedure described for the synthesis of compound 58 used for the synthesis of compound 82 (87%). HRMS (ESI+) Calcd for C.sub.164H.sub.181O.sub.34N.sub.2SiP.sup.+ [M-Et.sub.3N].sup.+ 2782.2036. found 2782.2077.

Synthesis of 83

[0846] ##STR00269##

[0847] The procedure described for the synthesis of compound 59 used for the synthesis of compound 83 (88%). HRMS (ESI+) Calcd for C.sub.169H.sub.190O.sub.35N.sub.5SiP.sup.+ [M-Et.sub.3N].sup.+ 2910.2815. found 2910.2841.

Synthesis of 84

[0848] ##STR00270##

[0849] The procedure described for the synthesis of compound 60 used for the synthesis of compound 84 (90%). HRMS (ESI+) Calcd for C.sub.153H.sub.172O.sub.35N.sub.5P.sup.+ [M-Et.sub.3N].sup.+ 2672.1638. found 2672.1759.

Synthesis of 33

[0850] ##STR00271##

[0851] The procedure described for the synthesis of compound 33 from 32 used for the synthesis of compound 33 (55%). HRMS (ESI+) Calcd for C.sub.46H.sub.82N.sub.3PO.sub.34.sup.+ [M-Na+2H].sup.+ 1252.4551. found 1252.4574.

Synthesis of 85

[0852] ##STR00272##

[0853] To a solution of 84 in DCM, were added dibenzyl N,N-diisopropylphosphoramidite (2.0 equiv.) and diisopropylammonium tetrazolide (1.5 equiv.) and the solution was stirred at rt for 2.5 h. Then, t-butyl peroxide (6.0 equiv., 5.0-6.0 M solution in decane) was added and the reaction mixture stirred for 1 h. After 1 h, reaction mixture was diluted with DCM and quenched with NaHCO.sub.3 aq. sat. solution. The aqueous layer was extracted with DCM. The combined organic layer was washed with brine, dried over Na.sub.2SO.sub.4, filtered, and the filtrate was concentrated under vacuum to obtain the crude product. The crude product was purified by automated flash column chromatography on silica gel (0-100% EtOAc in cyclohexane) to afford the desired product 85 (88%). HRMS (ESI+) Calcd for C.sub.167H.sub.185O.sub.38N.sub.5P.sub.2.sup.+ [M-Et.sub.3N].sup.+ 2932.2240. found 2932.2147.

Synthesis of 54

[0854] ##STR00273##

[0855] Pd/C (20 mg) was added to a clear solution of 85 (20 mg) in EtOAc:MeOH:H.sub.2O:DCM. Obtained inhomogeneous mixture was stirred under hydrogen atmosphere at rt for 40 h. After 40 h, reaction mixture was filtered through PTFE filter and concentrated under vacuum at 30° C. bath temperature of rotary evaporator for 10 min to remove methanol, EtOAc, DCM and water. The crude product obtained after solvents removal was dissolved in MeOH, water and to this LiOH (2 N in water) was added at 0° C. The reaction mixture was stirred at 0° C. for 3 h. After 3 h, the reaction mixture was quenched with AcOH and the solvents were removed under reduced pressure and the obtained crude residue was purified with C18 reverse phase column chromatography using water and acetonitrile as solvents to give the desired final compound 54 in salt form. Then triethylamine salt was exchanged with Dowex resin to give the desired compound with sodium salt. (40% over 3 steps) as a white solid. HRMS (ESI+) Calcd for C.sub.46H.sub.83N.sub.3P.sub.2O.sub.37.sup.+ [M−Na+H].sup.+ 1332.4214. found 1332.4242.

Synthesis of 86

[0856] ##STR00274##

[0857] The procedure described for the synthesis of compound 58 used for the synthesis of compound 86 (94%). HRMS (ESI+) Calcd for C.sub.165H.sub.203O.sub.37N.sub.7P.sub.2.sup.+ [M-2×Et.sub.3N+H].sup.+ 2735.1318. found 2735.1356.

Synthesis of 87

[0858] ##STR00275##

[0859] H-phosphonate 86 (1.0 equiv.) and benzyl alcohol (10.0 equiv.) were co-evaporated with pyridine and dried under vacuum for 30 min. After that, it was dissolved in py and to this PivCl (5.0 equiv.) was added. The reaction mixture was kept for stirring at rt for 2 h. After 2 h, the reaction was cooled to −40° C., a freshly prepared solution of I.sub.2 in Py:H.sub.2O (20:1) was added and the reaction mixture was kept for stirring at the same temperature for 1.5 h and later brought to rt and stirred at rt for 15 min. Then, TEAB (10 mL) was added to the mixture and diluted with dichloromethane, washed successively with 10% aq. sodium thiosulfate, 1 M aq. triethylammonium hydrogen carbonate (TEAB), dried over Na.sub.2SO.sub.4, filtered and concentrated. The residue was purified by automated flash column chromatography (ethyl acetate:DCM:MeOH) together with 2% trimethylamine as eluents give the desired product 87 (86%) as viscous liquid. Maldi (ESI+) Calcd for C.sub.160H.sub.179N.sub.5P.sub.2O.sub.38.sup.+ [M+H-2×Et.sub.3N].sup.+ 2842.1770. found 2842.1638.

Synthesis of 54

[0860] ##STR00276##

[0861] Pd/C (20 mg) was added to a clear solution of 87 (20 mg) in EtOAc:MeOH:H.sub.2O:DCM. Obtained inhomogeneous mixture was stirred under hydrogen atmosphere at rt for 40 h. After 40 h, reaction mixture was filtered through PTFE filter and concentrated under vacuum at 30° C. bath temperature of rotary evaporator for 10 min to remove methanol, EtOAc, DCM and water. The crude product obtained after solvents removal was dissolved in MeOH, water and to this LiOH (2 N in water) was added at 0° C. The reaction mixture was stirred at 0° C. for 3 h. After 3 h, the reaction mixture was quenched with AcOH and the solvents were removed under reduced pressure and the obtained crude residue was purified with C18 reverse phase column chromatography using water and acetonitrile as solvents to give the desired final compound 54 in salt form. Then triethylamine salt was exchanged with Dowex resin to give the desired compound with sodium salt. (70% over 3 steps) as a white solid. HRMS (ESI+) Calcd for C.sub.46H.sub.83N.sub.3P.sub.2O.sub.37.sup.+ [M-3Na+4H].sup.+ 1332.4214. found 1332.4232.

A.6 Synthesis of Dodecasaccharide 92

Synthesis of 88

[0862] ##STR00277##

[0863] The procedure described for the synthesis of compound 32 was used for the synthesis of compound 88, here the only change is, in second step instead of a linker compound 52 was used as nucleophile.

Synthesis of 89

[0864] ##STR00278##

[0865] The procedure described for the synthesis of compound 52 used for the synthesis of compound 89.

Synthesis of 90

[0866] ##STR00279##

[0867] The procedure described for the synthesis of compound 33 used for the synthesis of compound 90.

Synthesis of 91

[0868] ##STR00280##

[0869] The procedure described for the synthesis of compound 53 used for the synthesis of compound 91.

Synthesis of 92

[0870] ##STR00281##

[0871] The procedure described for the synthesis of compound 33 used for the synthesis of compound 92 (60%). HRMS (ESI+) Calcd for C.sub.87H.sub.151N.sub.5P.sub.2O.sub.67 [(M−2Na+2H)/2] 1199.9019. found 1199.8950.

Conjugation of 92 with CRM.sub.197 and BSA

##STR00282## ##STR00283## ##STR00284##

[0872] The procedure described for the synthesis of glycoconjugates 36 and 37 was used for the synthesis of 94 and 95.

A.7 Alternative Synthesis of Dodecasaccharide 92

Synthesis of 96

[0873] ##STR00285##

[0874] H-phosphonate 58 (1.2 equiv.) and acceptor 60 (1.0 equiv.) were co-evaporated with pyridine and dried under vacuum for 30 min. After that, it was dissolved in py and to this PivCl (1.3 equiv.) was added. The reaction mixture was kept for stirring at rt for 3 h. After 3 h, the reaction was cooled to −40° C., a freshly prepared solution of I.sub.2 in Py:H.sub.2O (250 μL, 20:1) was added and the reaction mixture was kept for stirring at the same temperature for 1.5 h and later brought to rt and stirred at rt for 15 min. Then, TEAB (10 mL) was added to the mixture and diluted with dichloromethane, washed successively with 10% aq. sodium thiosulfate, 1 M aq. triethylammonium hydrogen carbonate (TEAB), dried over Na.sub.2SO.sub.4, filtered and concentrated. The residue was purified by automated flash column chromatography (ethyl acetate:DCM:MeOH) together with 2% trimethylamine as eluents give the desired product 96 (70%) as viscous liquid. MALDI (ESI+) Calcd for C.sub.287H.sub.325K.sub.2N.sub.7O.sub.75P.sub.2Si.sup.+ [M−2Et.sub.3N+2K].sup.+ 5237.0351. found 5237.718.

Synthesis of 97

[0875] ##STR00286##

[0876] The procedure described for the synthesis of compound 60 was used for the synthesis of compound 97 (70%). Maldi (ESI+) Calcd for C.sub.271H.sub.309N.sub.7O.sub.75P.sub.2.sup.+ [M].sup.+ 4926.3745. found 4926.323.

Synthesis of 92

[0877] ##STR00287## ##STR00288##

[0878] The procedure described for the synthesis of compound 61 was used for the synthesis of compound 98 (89%). The procedure described for the synthesis of compound 54 was used for the synthesis of compound 92 (60%). HRMS (ESI+) Calcd for C.sub.87H.sub.152N.sub.5P.sub.3NH.sub.4O.sub.70.sup.−[(M+NH.sub.4-2H)/2].sup.− 1247.8944. found 1247.8791.

A.8 Synthesis of Hexasaccharide 112

Synthesis of 99

[0879] ##STR00289##

[0880] MsCl and pyridine (py) were added to a clear solution of 8 in DCM at 0° C. The reaction mixture was stirred at room temperature overnight and then diluted with DCM, washed with aq. NaHCO.sub.3 solution, dried over Na.sub.2SO.sub.4 and concentrated to give the crude product. The residue was purified by automated silica gel chromatography (hexane/AcOEt) to give compound 99.

Synthesis of 100 and 101

[0881] ##STR00290##

[0882] Sodium iodide was added to a clear solution of 99 in 2-butanone and the reaction mixture was stirred at 100° C. for overnight. Then, the solvent was removed, and the crude residue was dissolved in DCM, washed with aq. NaHSO.sub.3, dried over Na.sub.2SO.sub.4 and concentrated to give the iodomethyl derivative 100. This iodo derivative was dissolved in freshly distilled trimethylphosphite and the solution was heated to 100° C. under vacuum (water pump) for 48 h. After concentration and silica gel chromatography phosphonate derivative 101 was obtained.

Synthesis of 102

[0883] ##STR00291##

[0884] TEA and thiophenol were added to a clear solution of 101 in THF. The reaction mixture was stirred at room temperature for 24 h. After complete consumption of starting material, the reaction mixture was diluted with TEA and concentrated to give a crude residue, and it was purified by silica gel chromatography to give 102.

Synthesis of 103

[0885] ##STR00292##

[0886] Phosphonate 102, linker and triphenylphosphine were dissolved in THF and the solution was cooled at 0° C. and to this DIAD was added. The mixture was stirred at room temperature for 24 h. After 24 h, the solution was concentrated and crude product was purified by silica gel chromatography to give 103.

Synthesis of 104

[0887] ##STR00293##

[0888] TEA and thiophenol were added to a clear solution of 103 in THF. The reaction mixture was stirred at room temperature for 24 h. After complete consumption of starting material, the reaction mixture was diluted with TEA and concentrated to give a crude residue, and it was purified by silica gel chromatography to give 104.

Synthesis of 105

[0889] ##STR00294##

[0890] Phosphonate derivative 104 was dissolved in 0.05 M solution of NaOMe in MeOH and stirred at rt for 10 min. Then reaction mixture was quenched with AcOH and the solvents were removed under vacuum. The obtained crude residue was purified by silica gel chromatography to give 105.

Synthesis of 106

[0891] ##STR00295##

[0892] Reaction was performed in accordance with the synthesis of compound 16.

Synthesis of 107

[0893] ##STR00296##

[0894] Reaction was performed in accordance with the synthesis of compound 18.

Synthesis of 108

[0895] ##STR00297##

[0896] Reaction was performed in accordance with the synthesis of compound 19.

Synthesis of 109

[0897] ##STR00298##

[0898] Reaction was performed in accordance with the synthesis of compound 28.

Synthesis of 110

[0899] ##STR00299##

[0900] Reaction was performed in accordance with the synthesis of compound 29.

Synthesis of 111

[0901] ##STR00300##

[0902] Reaction was performed in accordance with the synthesis of compound 30.

Synthesis of 112

[0903] ##STR00301##

[0904] Reaction was performed in accordance with the synthesis of compound 33.

Conjugation of 112 with CRM197 or BSA

##STR00302##

[0905] Reaction was performed in accordance with the conjugation of compound 33.

A.8 Synthesis of Hexasaccharide 117

Synthesis of 116

[0906] ##STR00303##

[0907] H-phosphonate 58 and linker were co-evaporated with pyridine and dried under vacuum for 30 min. After that, it was dissolved in pyridine and to this PivCl was added. The reaction mixture was kept for stirring at r.t. for 2 h. After 2 h, the reaction was cooled to −40° C., a freshly prepared solution of I.sub.2 in pyridine:H.sub.2O (20:1) was added and the reaction mixture was kept for stirring at the same temperature for 1.5 h and later brought to rt and stirred at rt for 15 min. Then, TEAB (10 mL) was added to the mixture and diluted with dichloromethane, washed successively with 10% aq. sodium thiosulfate, 1 M aq. triethylammonium hydrogen carbonate (TEAB), dried over Na.sub.2SO.sub.4, filtered and concentrated. The residue was purified by automated flash column chromatography (ethyl acetate:DCM:MeOH) together with 2% trimethylamine as eluents give the desired product 116 as viscous liquid.

Synthesis of 117

[0908] ##STR00304##

[0909] Reaction was performed in accordance with the synthesis of compound 31.

Conjugation of 117 with CRM.sub.197 or BSA

##STR00305##

[0910] Reaction was performed in accordance with the conjugation of compound 33.

A.9 Synthesis of Hexasaccharide 122

Synthesis of 121

[0911] ##STR00306##

[0912] H-phosphonate 58 and linker were co-evaporated with pyridine and dried under vacuum for 30 min. After that, it was dissolved in pyridine and to this PivCl was added. The reaction mixture was kept for stirring at r.t. for 2 h. After 2 h, the reaction was cooled to −40° C., a freshly prepared solution of I.sub.2 in pyridine:H.sub.2O (20:1) was added and the reaction mixture was kept for stirring at the same temperature for 1.5 h and later brought to rt and stirred at rt for 15 min. Then, TEAB (10 mL) was added to the mixture and diluted with dichloromethane, washed successively with 10% aq. sodium thiosulfate, 1 M aq. triethylammonium hydrogen carbonate (TEAB), dried over Na.sub.2SO.sub.4, filtered and concentrated. The residue was purified by automated flash column chromatography (ethyl acetate:DCM:MeOH) together with 2% trimethylamine as eluents give the desired product 121 as viscous liquid.

Synthesis of 122

[0913] ##STR00307##

[0914] Reaction was performed in accordance with the synthesis of compound 33 and a TBS deprotection step.

Conjugation of 122 with CRM.sub.197 or BSA

##STR00308##

[0915] Reaction was performed in accordance with the conjugation of compound 33.

A.10 Synthesis of Hexasaccharide 127

Synthesis of 126

[0916] ##STR00309##

[0917] H-phosphonate 58 and linker were co-evaporated with pyridine and dried under vacuum for 30 min. After that, it was dissolved in pyridine and to this PivCl was added. The reaction mixture was kept for stirring at r.t. for 2 h. After 2 h, the reaction was cooled to −40° C., a freshly prepared solution of I.sub.2 in pyridine:H.sub.2O (20:1) was added and the reaction mixture was kept for stirring at the same temperature for 1.5 h and later brought to rt and stirred at rt for 15 min. Then, TEAB (10 mL) was added to the mixture and diluted with dichloromethane, washed successively with 10% aq. sodium thiosulfate, 1 M aq. triethylammonium hydrogen carbonate (TEAB), dried over Na.sub.2SO.sub.4, filtered and concentrated. The residue was purified by automated flash column chromatography (ethyl acetate:DCM:MeOH) together with 2% trimethylamine as eluents give the desired product 126 as viscous liquid.

Synthesis of 127

[0918] ##STR00310##

[0919] Reaction was performed in accordance with the synthesis of compound 33 and a TBS deprotection step.

Conjugation of 127 with CRM.sub.197 or BSA

##STR00311##

[0920] Reaction was performed in accordance with the conjugation of compound 33.

A.11 Synthesis of Hexasaccharide 132

Synthesis of 131

[0921] ##STR00312##

[0922] H-phosphonate 58 and linker were co-evaporated with pyridine and dried under vacuum for 30 min. After that, it was dissolved in pyridine and to this PivCl was added. The reaction mixture was kept for stirring at r.t. for 2 h. After 2 h, the reaction was cooled to −40° C., a freshly prepared solution of I.sub.2 in pyridine:H.sub.2O (20:1) was added and the reaction mixture was kept for stirring at the same temperature for 1.5 h and later brought to rt and stirred at rt for 15 min. Then, TEAB (10 mL) was added to the mixture and diluted with dichloromethane, washed successively with 10% aq. sodium thiosulfate, 1 M aq. triethylammonium hydrogen carbonate (TEAB), dried over Na.sub.2SO.sub.4, filtered and concentrated. The residue was purified by automated flash column chromatography (ethyl acetate:DCM:MeOH) together with 2% trimethylamine as eluents give the desired product 131 as viscous liquid.

Synthesis of 132

[0923] ##STR00313##

[0924] Reaction was performed in accordance with the synthesis of compound 33 and a TBS deprotection step.

Conjugation of 132 with CRM.sub.197 or BSA

##STR00314##

[0925] Reaction was performed in accordance with the conjugation of compound 33.

A.12 Synthesis of Hexasaccharide 137

Synthesis of 136

[0926] ##STR00315##

[0927] H-phosphonate 58 and linker were co-evaporated with pyridine and dried under vacuum for 30 min. After that, it was dissolved in pyridine and to this PivCl was added. The reaction mixture was kept for stirring at r.t. for 2 h. After 2 h, the reaction was cooled to −40° C., a freshly prepared solution of I.sub.2 in pyridine:H.sub.2O (20:1) was added and the reaction mixture was kept for stirring at the same temperature for 1.5 h and later brought to rt and stirred at rt for 15 min. Then, TEAB (10 mL) was added to the mixture and diluted with dichloromethane, washed successively with 10% aq. sodium thiosulfate, 1 M aq. triethylammonium hydrogen carbonate (TEAB), dried over Na.sub.2SO.sub.4, filtered and concentrated. The residue was purified by automated flash column chromatography (ethyl acetate:DCM:MeOH) together with 2% trimethylamine as eluents give the desired product 136 as viscous liquid.

Synthesis of 137

[0928] ##STR00316##

[0929] Reaction was performed in accordance with the synthesis of compound 33 and a TBS deprotection step.

Conjugation of 137 with CRM.sub.197 or BSA

##STR00317##

[0930] Reaction was performed in accordance with the conjugation of compound 33.

A.13 Synthesis of Hexasaccharide 142

Synthesis of 141

[0931] ##STR00318##

[0932] H-phosphonate 58 and linker were co-evaporated with pyridine and dried under vacuum for 30 min. After that, it was dissolved in pyridine and to this PivCl was added. The reaction mixture was kept for stirring at r.t. for 2 h. After 2 h, the reaction was cooled to −40° C., a freshly prepared solution of I.sub.2 in pyridine:H.sub.2O (20:1) was added and the reaction mixture was kept for stirring at the same temperature for 1.5 h and later brought to rt and stirred at rt for 15 min. Then, TEAB (10 mL) was added to the mixture and diluted with dichloromethane, washed successively with 10% aq. sodium thiosulfate, 1 M aq. triethylammonium hydrogen carbonate (TEAB), dried over Na.sub.2SO.sub.4, filtered and concentrated. The residue was purified by automated flash column chromatography (ethyl acetate:DCM:MeOH) together with 2% trimethylamine as eluents give the desired product 141 as viscous liquid.

Synthesis of 142

[0933] ##STR00319##

[0934] Reaction was performed in accordance with the synthesis of compound 33 and a TBS deprotection step.

Conjugation of 142 with CRM.sub.197 or BSA

##STR00320##

[0935] Reaction was performed in accordance with the conjugation of compound 33.

A.14 Synthesis of Hexasaccharide 147

Synthesis of 146

[0936] ##STR00321##

[0937] H-phosphonate 58 and linker were co-evaporated with pyridine and dried under vacuum for 30 min. After that, it was dissolved in pyridine and to this PivCl was added. The reaction mixture was kept for stirring at r.t. for 2 h. After 2 h, the reaction was cooled to −40° C., a freshly prepared solution of I.sub.2 in pyridine:H.sub.2O (20:1) was added and the reaction mixture was kept for stirring at the same temperature for 1.5 h and later brought to rt and stirred at rt for 15 min. Then, TEAB (10 mL) was added to the mixture and diluted with dichloromethane, washed successively with 10% aq. sodium thiosulfate, 1 M aq. triethylammonium hydrogen carbonate (TEAB), dried over Na.sub.2SO.sub.4, filtered and concentrated. The residue was purified by automated flash column chromatography (ethyl acetate:DCM:MeOH) together with 2% trimethylamine as eluents give the desired product 146 as viscous liquid.

Synthesis of 142

[0938] ##STR00322##

[0939] Reaction was performed in accordance with the synthesis of compound 33 and a TBS deprotection step.

Conjugation of 147 with CRM.sub.197 or BSA

##STR00323##

[0940] Reaction was performed in accordance with the conjugation of compound 33.

A.15 Synthesis of Hexasaccharide 152

Synthesis of 151

[0941] ##STR00324##

[0942] H-phosphonate 58 and linker were co-evaporated with pyridine and dried under vacuum for 30 min. After that, it was dissolved in pyridine and to this PivCl was added. The reaction mixture was kept for stirring at r.t. for 2 h. After 2 h, the reaction was cooled to −40° C., a freshly prepared solution of I.sub.2 in pyridine:H.sub.2O (20:1) was added and the reaction mixture was kept for stirring at the same temperature for 1.5 h and later brought to rt and stirred at rt for 15 min. Then, TEAB (10 mL) was added to the mixture and diluted with dichloromethane, washed successively with 10% aq. sodium thiosulfate, 1 M aq. triethylammonium hydrogen carbonate (TEAB), dried over Na.sub.2SO.sub.4, filtered and concentrated. The residue was purified by automated flash column chromatography (ethyl acetate:DCM:MeOH) together with 2% trimethylamine as eluents give the desired product 151 as viscous liquid.

Synthesis of 152

[0943] ##STR00325##

[0944] Reaction was performed in accordance with the synthesis of compound 33 and a TBS deprotection step.

Conjugation of 152 with CRM.sub.197 or BSA

##STR00326##

[0945] SBAP (N-succinimidyl-3-(bromoacetamido)propionate) was added to a stirred solution of protein in sodium phosphate buffer (NaPi, pH 7.4) at room temperature. The reaction mixture was stirred for one hour at room temperature and afterwards concentrated using membrane filtration and rebuffered in NaPi (pH 8.0). A solution of compound 152 in NaPi was added to the solution of activated protein and stirred at r.t. for 16 hours. The glycoconjugate was then washed with sterile water and treated with 1-cysteine in sterile water. Purification of the glycoconjugate was achieved by membrane filtration.

A.16 Synthesis of Hexasaccharide 157

Synthesis of 156

[0946] ##STR00327##

[0947] H-phosphonate 58 and linker were co-evaporated with pyridine and dried under vacuum for 30 min. After that, it was dissolved in pyridine and to this PivCl was added. The reaction mixture was kept for stirring at r.t. for 2 h. After 2 h, the reaction was cooled to −40° C., a freshly prepared solution of I.sub.2 in pyridine:H.sub.2O (20:1) was added and the reaction mixture was kept for stirring at the same temperature for 1.5 h and later brought to rt and stirred at rt for 15 min. Then, TEAB (10 mL) was added to the mixture and diluted with dichloromethane, washed successively with 10% aq. sodium thiosulfate, 1 M aq. triethylammonium hydrogen carbonate (TEAB), dried over Na.sub.2SO.sub.4, filtered and concentrated. The residue was purified by automated flash column chromatography (ethyl acetate:DCM:MeOH) together with 2% trimethylamine as eluents give the desired product 156 as viscous liquid.

Synthesis of 157

[0948] ##STR00328##

[0949] Reaction was performed in accordance with the synthesis of compound 33 and a TBS deprotection step.

Conjugation of 157 with CRM.sub.197 or BSA

##STR00329##

[0950] Reaction was performed in accordance with the conjugation of compound 33.

A.17 Synthesis of Octadecasaccharides 162, 163, 164 and 165

Synthesis of 161

[0951] ##STR00330##

[0952] The procedure described for the synthesis of compound 32 was used for the synthesis of compound 161, here with the only change that in the second step instead of a linker compound 89 was used as nucleophile.

Synthesis of 162

[0953] ##STR00331##

[0954] wherein Z represents

##STR00332##

[0955] Compound 162 was synthesized from compound 161 as described for compound 89 (removal of the TBDPS protecting group) and thereafter as described for compound 90.

Synthesis of 163

[0956] ##STR00333##

[0957] wherein Z represents

##STR00334##

[0958] Compound 163 was synthesized from compound 161 as described for compound 89 (removal of the TBDPS protecting group) and thereafter as described for compounds 91 and 92.

Synthesis of 164

[0959] ##STR00335##

[0960] wherein Z represents

##STR00336##

[0961] The phosphonate compound 164 was synthesized as described for compound 162.

Synthesis of 165

[0962] ##STR00337##

[0963] wherein Z represents

##STR00338##

[0964] The phosphonate compound 165 was synthesized as described for compound 163.

A.18 Alternative Synthesis of Octadecasaccharides 162 and 163

Synthesis of 166

[0965] ##STR00339##

[0966] The procedure described for the synthesis of compound 86 used for the synthesis of compound 166.

Synthesis of 167

[0967] ##STR00340##

[0968] The procedure described for the synthesis of compound 96 used for the synthesis of compound 167.

Synthesis of 162

[0969] ##STR00341##

[0970] Compound 162 was synthesized from compound 167 as described for compound 33.

Synthesis of 168

[0971] ##STR00342##

[0972] The procedure described for the synthesis of compound 60 used for the synthesis of compound 168.

Synthesis of 169

[0973] ##STR00343##

[0974] The procedure described for the synthesis of compound 86 used for the synthesis of compound 169.

Synthesis of 170

[0975] ##STR00344##

[0976] The procedure described for the synthesis of compound 87 used for the synthesis of compound 170.

Synthesis of 163

[0977] ##STR00345##

[0978] Compound 163 was synthesized from compound 170 as described for compound 54.

A.19 Synthesis of Tetracosasaccharides 172 and 173

Synthesis of 172

[0979] ##STR00346##

[0980] wherein Z represents

##STR00347##

[0981] Compound 172 was synthesized from dodecasaccharide 89 which was attached to the dodecasaccharide 171

##STR00348##

[0982] according to the procedure described for compound 88 following deprotection of the TBDPS group as described for compound 89 and subsequently complete deprotection as described for compound 90.

Synthesis of 173

[0983] ##STR00349##

[0984] wherein Z represents

##STR00350##

[0985] Compound 173 was synthesized from the dodecasaccharide 89 which was attached to the dodecasaccharide 171 according to the procedure described for compound 88 following deprotection of the TBDPS group as described for compound 89, phosphorylation as described for compound 91 and subsequently complete deprotection as described for compound 92.

A.20 Alternative Synthesis of Tetracosasaccharides 172 and 173

Synthesis of 174

[0986] ##STR00351##

[0987] The procedure described for the synthesis of compound 96 used for the synthesis of compound 174.

Synthesis of 172

[0988] ##STR00352##

[0989] Compound 172 was synthesized from compound 174 as described for compound 33.

Synthesis of 175

[0990] ##STR00353##

[0991] The procedure described for the synthesis of compound 60 used for the synthesis of compound 175.

Synthesis of 176

[0992] ##STR00354##

[0993] The procedure described for the synthesis of compound 86 used for the synthesis of compound 176.

Synthesis of 177

[0994] ##STR00355##

[0995] The procedure described for the synthesis of compound 87 used for the synthesis of compound 177.

Synthesis of 173

[0996] ##STR00356##

[0997] Compound 173 was synthesized from compound 177 as described for compound 54.

A.21 Synthesis of Triacontasaccharides 179 and 183

Synthesis of 178

[0998] ##STR00357##

[0999] The procedure described for the synthesis of compound 96 used for the synthesis of compound 178.

Synthesis of 179

[1000] ##STR00358##

[1001] Compound 179 was synthesized from compound 178 as described for compound 33.

Synthesis of 180

[1002] ##STR00359##

[1003] The procedure described for the synthesis of compound 60 used for the synthesis of compound 180.

Synthesis of 181

[1004] ##STR00360##

[1005] The procedure described for the synthesis of compound 86 used for the synthesis of compound 181.

Synthesis of 182

[1006] ##STR00361##

[1007] The procedure described for the synthesis of compound 87 used for the synthesis of compound 182.

Synthesis of 183

[1008] ##STR00362##

[1009] Compound 183 was synthesized from compound 182 as described for compound 54.

A.22 Synthesis of Hexatriacontasasaccharides 186 and 187

Synthesis of 185

[1010] ##STR00363##

[1011] wherein Z represents

##STR00364##

[1012] Compound 185 was synthesized from octadecasaccharide 161 from which the TBDPS protecting group was selectively removed according to the procedure described for compound 89. Thereafter the TBDPS deprotected trisaccharide was reacted with compound 184

##STR00365##

[1013] in order to obtain the saccharide 185.

Synthesis of 186

[1014] ##STR00366##

[1015] wherein Z represents

##STR00367##

[1016] Compound 186 was synthesized from saccharide 185 which was converted according to the procedures described for compound 89 (removal of the TBDPS protecting group) and thereafter for compound 90 (removal of the TBDPS protecting group).

Synthesis of 187

[1017] ##STR00368##

[1018] wherein Z represents

##STR00369##

[1019] Compound 187 was synthesized from saccharide 185 which was converted according to the procedures described for compound 89 (removal of the TBDPS protecting group), phosphorylation as described for compound 91 and subsequently complete deprotection as described for compound 92.

A.23 Alternative Synthesis of Hexatriacontasasaccharides 186 and 187

Synthesis of 188

[1020] ##STR00370##

[1021] The procedure described for the synthesis of compound 96 used for the synthesis of compound 188.

Synthesis of 186

[1022] ##STR00371##

[1023] Compound 186 was synthesized from compound 188 as described for compound 33.

Synthesis of 189

[1024] ##STR00372##

[1025] The procedure described for the synthesis of compound 60 used for the synthesis of compound 189.

Synthesis of 190

[1026] ##STR00373##

[1027] The procedure described for the synthesis of compound 86 used for the synthesis of compound 190.

Synthesis of 191

[1028] ##STR00374##

[1029] The procedure described for the synthesis of compound 87 used for the synthesis of compound 191.

Synthesis of 187

[1030] ##STR00375##

[1031] Compound 187 was synthesized from compound 191 as described for compound 54.

A.24 Synthesis of Oligosaccharides 193 and 197

Synthesis of 192

[1032] ##STR00376##

[1033] The procedure described for the synthesis of compound 96 used for the synthesis of compound 192.

Synthesis of 193

[1034] ##STR00377##

[1035] Compound 193 was synthesized from compound 192 as described for compound 33.

Synthesis of 194

[1036] ##STR00378##

[1037] The procedure described for the synthesis of compound 60 used for the synthesis of compound 194.

Synthesis of 195

[1038] ##STR00379##

[1039] The procedure described for the synthesis of compound 86 used for the synthesis of compound 195.

Synthesis of 196

[1040] ##STR00380##

[1041] The procedure described for the synthesis of compound 87 used for the synthesis of compound 196.

Synthesis of 197

[1042] ##STR00381##

[1043] Compound 197 was synthesized from compound 196 as described for compound 54.

A.25 Synthesis of Oligosaccharides 199 and 203

Synthesis of 198

[1044] ##STR00382##

[1045] The procedure described for the synthesis of compound 96 used for the synthesis of compound 198.

Synthesis of 199

[1046] ##STR00383##

[1047] Compound 199 was synthesized from compound 198 as described for compound 33.

Synthesis of 200

[1048] ##STR00384##

[1049] The procedure described for the synthesis of compound 60 used for the synthesis of compound 200.

Synthesis of 201

[1050] ##STR00385##

[1051] The procedure described for the synthesis of compound 86 used for the synthesis of compound 201.

Synthesis of 202

[1052] ##STR00386##

[1053] The procedure described for the synthesis of compound 87 used for the synthesis of compound 202.

Synthesis of 203

[1054] ##STR00387##

[1055] Compound 203 was synthesized from compound 202 as described for compound 54.

A.26 Synthesis of Oligosaccharides 205 and 209

Synthesis of 204

[1056] ##STR00388## ##STR00389##

[1057] The procedure described for the synthesis of compound 96 used for the synthesis of compound 204.

Synthesis of 205

[1058] ##STR00390##

[1059] Compound 205 was synthesized from compound 204 as described for compound 33.

Synthesis of 206

[1060] ##STR00391##

[1061] The procedure described for the synthesis of compound 60 used for the synthesis of compound 206.

Synthesis of 207

[1062] ##STR00392##

[1063] The procedure described for the synthesis of compound 86 used for the synthesis of compound 207.

Synthesis of 208

[1064] ##STR00393##

[1065] The procedure described for the synthesis of compound 87 used for the synthesis of compound 208.

Synthesis of 209

[1066] ##STR00394##

[1067] Compound 209 was synthesized from compound 208 as described for compound 54.

A.27 Synthesis of Oligosaccharides 211 and 215

Synthesis of 210

[1068] ##STR00395##

[1069] The procedure described for the synthesis of compound 96 used for the synthesis of compound 210.

Synthesis of 211

[1070] ##STR00396##

[1071] Compound 211 was synthesized from compound 210 as described for compound 33.

Synthesis of 212

[1072] ##STR00397##

[1073] The procedure described for the synthesis of compound 60 used for the synthesis of compound 212.

Synthesis of 213

[1074] ##STR00398##

[1075] The procedure described for the synthesis of compound 86 used for the synthesis of compound 213.

Synthesis of 214

[1076] ##STR00399##

[1077] The procedure described for the synthesis of compound 87 used for the synthesis of compound 214.

Synthesis of 215

[1078] ##STR00400##

[1079] Compound 215 was synthesized from compound 214 as described for compound 54.

B. Stability Studies

[1080] Cleavage of the Phosphate Bond in Compound 33 with NaOH

[1081] Next the stability of the compounds of the present invention was tested and assessed. The task was to find out how stable are compounds 33, 54, 90, 92, 112, 117, 162, 163, 164, 165, 172, and 173 under formulation conditions. Prior to the stability in Alhydrogel, PBS buffer and water, the compound 33 was treated with 0.1 M sodium hydroxide at room temperature. Here it was found that compound 33 is cleaving very slowly only under highly basic conditions. However, even after 4 days (10 μg of 33 in 200 μL) under these drastic conditions, only 50% of compound 33 was cleaved and still 50% of compound 33 was observed being intact in HPLC chromatogram (FIGS. 6 and 7).

Stability of Compound 33 Over Alhydrogel in PBS, PBS and Water:

[1082] Next the stability of the compound 33 under formulation conditions was scrutinized. Each formulation vial contains, 30 μg of 33 in i) Alhydrogel in PBS or ii) PBS alone or iii) water (overall volume of the solution is 500 μL). NaPi is used as a synonym for PBS herein. 60 μL of Alhydrogel containing 0.6 mg of Aluminium were used for each experiment. These three formulated solutions were kept at 37° C., 25° C. and 2-8° C. for 14 days. After every 24 h duration, 50 μL of the solution from each vial i) Alhydrogel in PBS, ii) PBS alone and iii) water at 37° C., 25° C. and 2-8° C. was aliquoted and analyzed by HPLC (FIGS. 8, 9, 10). From these studies it is evident that compound 33 is stable over the whole temperature range from 2° C. to 37° C. FIG. 8 shows the stability at 2-8° C. after 4 days, FIG. 9 at 2-8° C. after 14 days, FIG. 10 at 25° C. after 4 days, FIG. 11 at 25° C. after 14 days, FIG. 12 at 37° C. after 4 days, and FIG. 13 at 37° C. after 14 days.

[1083] In comparison to the natural polysaccharide PSII of Clostridium difficile the compounds 33, 54, 90, 92, 112, 117, 162, 163, 164, 165, 172, and 173 were found to be sufficiently stable under the formulation conditions described above.

[1084] It was also found that the natural polysaccharide PSII of Clostridium difficile composed of hexaglycosyl phosphate repeating units as shown below

##STR00401##

[1085] is not stable under NaOH treatment, not stable under acid conditions such as acetic acid and also not stable in solution at 2-8° C., 25° C. and 37° C. In was found that under these conditions the natural PSII degrades quickly to degradation products which no longer induce an immunological effect.

[1086] Therefore the stability experiments above demonstrate unambiguously that the compounds of the present invention are stable under conditions where the natural PSII decomposes to fragments no longer useful as vaccines, while the compounds disclosed herein are stable in solution and do not require to be lyophilized and re-dissolved, no cold storage, and do not require production and shipment applying an expensive working cold chain system.

C. Biological Experiments

SDS-PAGE Analysis.

[1087] The samples were mixed in a microfuge tube and heated for 5 min at 95° C. on a thermocycler. After cooling to room temperature for 5 min, the samples at approximately 2.5 μg were loaded onto the respective wells of a 10% polyacrylamide gel along with 10 μL of the marker. The samples were run at a constant voltage of 120 V for 1 h. Staining was done using the GelCode™ Blue Safe Protein Stain as per manufacture instructions. The gels were washed with deionized water overnight and scanned using the gel documentation system.

Size Exclusion Chromatography (SEC) of Glycoconjugates.

[1088] The glycoconjugates used for immunization studies were analyzed by SEC to observe a mass difference between the conjugated and unconjugated CRM protein. The samples were diluted in 50 mM Tris, 20 mM NaCl, pH 7.2 and run on an Agilent 1100 HPLC system fitted with Tosoh TSK G2000 column (SWxl, 7.8 mm×30 cm, 5 μm) and a Tosoh TSKgel® Guard Column (SWxl 6.0 mm×4 cm, 7 μm). The flow rate was kept at 1 mL/min.

Production of Glycoconjugate

[1089] The C. difficile PS-II synthetic antigens were conjugated to the carrier protein CRM.sub.197 for immunization experiments and to Bovine Serum Albumin (BSA) as coating antigen for ELISA (see A. Chemical Synthesis). The resulting conjugates were sterile filtered using a 0.2 μM membrane filter prior to use. The conjugates were analyzed by MALDI analysis. The loading of the saccharide on the carrier protein was specifically calculated by subtracting the mass between the conjugated and unconjugated protein using MALDI analysis. The protein content was estimated using the micro BCA method following manufacture protocol.

Characterization of Glycoconjugates 36 (33-CRM.sub.197), 56 (54-CRM.sub.197) and 94 (92-CRM.sub.197)

[1090] The C. difficile antigen glycoconjugates 36, 56 and 94 used for the immunization studies were analyzed for the conjugation efficiency and antigen content. MALDI-TOF MS analysis of the glycoconjugates revealed a good conjugation efficiency. The mass differences between the conjugated and unconjugated CRM.sub.197 protein yielded a loading of about 7.5 (56) and about 5 (94) antigens per CRM.sub.197 molecule.

[1091] The glycoconjugates were also analyzed by a 10% SDS-PAGE and SEC that revealed a clear mass shift as compared to the unconjugated CRM.sub.197 protein (FIGS. 24 and 25).

Immunization Studies

[1092] Study I—Immunological Evaluation of Semisynthetic Glycoconjugates of C. difficile Antigen PS-II Immunized in Rabbits.

1. Aim of the Study:

[1093] Evaluation of the IgG antibody response in rabbits immunized with C. difficile antigen PS-II semi-synthetic CRM.sub.197 conjugate vaccine 36.

2. Materials:

[1094] ELISA plates (high-binding, EIA/RIA Plate, 96 well, flat bottom with low evaporation lid, company: Costar® 3361) [1095] Detection antibody: Goat anti rabbit IgG peroxidase conjugate (Sigma, #A4914) [1096] Blocking solution: 1% FCS (v/v) in PBS [1097] Antibody diluent: PBS+1% BSA (w/v). [1098] Wash Buffer: PBS+0.1% Tween 20 (PBS-T) [1099] Developing solution: 1 Step™ Ultra TMB-ELISA developer. (ThermoScientific, Cat #: 34028) [1100] Stop solution: 2M Sulphuric acid (H.sub.2SO.sub.4) [1101] Plate reader: Anthos ht 2. [1102] Software: WinRead 2.36 for absorbance measurements and GraphPad Prism 7 for data plotting and analysis. [1103] Incomplete Freund's Adjuvant (IFA). InvivoGen; Cat: vac-ifa-10, Batch #: IFA-39-03; Exp Dt: September 2019 [1104] QuantiPro™ BCA Assay Kit (SIGMA) Product: QPBCA-1KT; Lot #: SLBR7451V; Pcode: 1002296464

3. Methods:

Formulation of Vaccines for Immunization

[1105] The C. difficile PS-II glycoconjugate 36 was formulated in Incomplete Freund's Adjuvant (IFA) for immunization in rabbits. Incomplete Freund's Adjuvant (IFA) from Invivogen was used for formulating the vaccines for rabbit immunization studies. Protocol was followed as per manufacture. Antigen: IFA concentration was kept at 1:1. The antigen dose per animal was kept at 2.5 μg/200 μL/animal (100 μL of antigen +100 μL IFA). IFA at the desired calculated volume (50% of the final immunization volume) was taken in a 15 mL sterile falcon. The calculated amount of the diluted antigen solution (Volume adjusted with PBS to 50% of the final immunization volume) was taken in a 3 mL sterile syringe, fitted with a 20 G needle. The DS solution was added into the falcon containing the IFA and immediately vortexed for 15 sec (5×). The color of the formulation changes from pale-yellow to milky-white on vortexing which indicates the formation of stable emulsion. The resulting vaccine formulation was briefly vortexed and aliquoted into 2 mL sterile tubes with the desired dose volumes. Prior to immunizations, the tubes containing the vaccine formulations were vortexed and then injected into animals.

Immunization Schedule

[1106] Rabbit immunizations were performed under specific pathogen-free conditions and were provided food and water ad libitum. Rabbits (n=4) were immunized sub cutaneous with the vaccine formulations at an injection volume of 200 μL/rabbit. The antigen dose for rabbit was kept at 2.5 μg/animal of PS-II antigen or corresponding volume of PBS for negative controls. Rabbits were immunized on day 0, 14 and 35. Blood was drawn on day 0, 7 and 42 for the determination of antibody titers.

4. Enzyme Linked Immunosorbent Assay (ELISA) of Sera Using In-House Antigen Coated Plates

[1107] Coating of Plates with Antigen

[1108] Antigen-BSA conjugates were used as the coating antigen. Antigen-BSA conjugates were dissolved at a concentration of 5 μg/mL in phosphate buffered saline (PBS) pH 7.4. 100 μL were coated per well and incubated overnight at 4° C. to get an antigen concentration of 0.5 μg/well.

Washing

[1109] After overnight adsorption of the antigen, the plates were washed 1× with PBS-T (200 μL/well) and the excess fluid per well was removed by inverting the plate and tapping on a clean dry tissue towel.

Blocking

[1110] The plates were blocked using 200 μL of the commercial blocking solution and incubated for 2h at RT.

Washing

[1111] After blocking, the plates were washed 3× with PBS-T (200 μL/well) and the excess fluid per well was removed by inverting the plate and by tapping on a clean dry tissue towel.

Dilution of Sera and Incubations

[1112] Pooled sera (n=4 rabbits) from different time-points of the different experimental groups were diluted to their respective dilutions in the antibody diluent (PBS+1% BSA). 100 μL of the diluted sera samples of the different experimental groups were added in duplicates to the corresponding wells and incubated on a shaker set at 250 rpm for 2h at RT. 100 μL/well of the antibody diluent (PBS+1% BSA) formed the experimental blank. After incubation with sera, the plates were washed 4× with PBS-T (200 μL/well) and the excess fluid per well was removed by inverting the plate and by tapping on a clean dry tissue towel.

Incubation (Detection Antibody)

[1113] The corresponding detection antibody, anti-rabbit IgG HRP conjugate was diluted 1:10,000 in the antibody diluent (PBS+1% BSA) and 100 μL/well was added and incubated on a shaker at 250 rpm for 1 h at RT. After the incubation with detection antibody, the plates were washed 5× with PBS-T (200 μL/well) and the excess fluid per well was removed by inverting the plate and by tapping on a clean dry tissue towel.

Substrate Addition

[1114] To each well, 100 μL of the ready to use TMB substrate (normalized to RT form 4° C.) was added and incubated in dark for 15 min. The blue color of the enzymatic reaction was stopped by adding 50 μL/well of 2M H.sub.2SO.sub.4 solution resulting in a yellow colored solution. The absorption of the yellow colored solution was measured at 450 nm using a plate reader.

Results

[1115] The absorption values were analyzed by plotting a graph using the Graphpad Prism software.

[1116] The ELISA data clearly show that sera from C. difficile PS-II conjugate 36 immunized rabbits recognize the corresponding antigens (see FIG. 15).

Study II—Immunological Evaluation of Semisynthetic Glycoconjugates of C. difficile Antigen PS-II Immunized in Rabbits and Mice.

1. Aim of the Study:

[1117] Evaluation of the IgG antibody response in rabbits and mice immunized with C. diff PS-II semi-synthetic CRM.sub.197 conjugate vaccines 56 and 94.

2. Materials:

[1118] ELISA plates (high-binding, EIA/RIA Plate, 96 well, flat bottom with low evaporation lid, company: Costar® 3361) [1119] Detection antibody: Goat anti rabbit IgG peroxidase conjugate (Sigma, #A4914) and anti-human IgG (H+L)-HRP, Nordic Immunology, Lot #:6276 [1120] Blocking solution: Roche, Ref: 11112589001; Lot: 21495200, Exp. Dt: July 2019. [1121] Antibody diluent: PBS+1% BSA (w/v) [1122] Wash Buffer: PBS+0.1% Tween 20 (PBS-T) [1123] Developing solution: 1 Step™ Ultra TMB-ELISA developer. (ThermoScientific, Cat #: 34028) [1124] Stop solution—2M Sulphuric acid (H.sub.2SO.sub.4) [1125] Plate reader: Anthos ht 2 [1126] Software: WinRead 2.36 for absorbance measurements and GraphPad Prism 7 for data plotting and analysis [1127] Alum: Aluminium Hydroxide Gel Adjuvant (Alhydrogel 2%), Brenntag, Batch #:5447 Exp Dt: February 2020 [1128] QuantiPro™ BCA Assay Kit (SIGMA) Product: QPBCA-1KT; Lot #: SLBR7451V; Pcode: 1002296464 [1129] Mini-PROTEAN® TGX™ Gels—10%, 10 well (30 μL/well) Control Nr: 64175708 [1130] Precision Plus Dual Color, Cat: 1610374; Control Nr: 641798899 [1131] Gel Code™ Blue Safe Protein Stain; ThermoScientific; Ref: 1860957; Lot #: TA260266 [1132] C. difficile coated ELISA plates for strains 630 (tgc BIOMICS Lot #: 630-43411) and R20291 (tgc BIOMICS Lot #: R20291-43559) Exp. Dt: May 2020. [1133] C. difficile positive patient plasma.

3. Methods

Formulation of Vaccines for Immunization in Aluminum Hydroxide (Alum) Adjuvant

[1134] All the formulations were prepared under sterile conditions. The glycoconjugates 56 and 94 (drug substances; DS) and PBS were mixed in the appropriate pre-calculated ratio in a 50 mL Falcon™ tube corresponding to the final formulation volume leaving out the volume of alum (0.25 mg/mL) required. This formed the DS-PBS mixture. The antigen/DS dose per animal was kept at 2.5 μg/500 μL/animal or 10 μg/500 μL/animal (rabbit studies) or at 0.5 μg/100 μL/animal or 2 μg/100 μL/animal (mouse studies). The DS-PBS mixtures were gently mixed (5×) using a serological pipette. To the DS-PBS mixtures, the corresponding volume of stock alum (10 mg/mL) was added to give a final alum ratio of 1:40 or 0.250 mg/mL. The mixtures were immediately mixed by gentle pipetting (20×) using a 5 mL serological pipette. The Falcon™ tubes were capped, wrapped with Parafilm® and allowed to mix on a shaker at 250 rpm for 2 h at room temperature (RT). After the incubation time of 2 h, the formulations were brought under the clean bench, aliquoted, and further stored at 4° C. until further use. The glycoconjugates formulated in Alum were characterized to determine the final alum concentration and the pH of the formulations.

Immunization Schedule

[1135] Mice and rabbit immunizations were performed under specific pathogen-free conditions and the animals were provided food and water ad libitum. Mice (n=7 or 8 per study arm) and rabbits (n=4 per study arm) were immunized subcutaneously with the vaccine formulations at an injection volume of 100 μL/mice, and 500 μL/rabbit with the different antigen doses. Mice were immunized on days 0, 14 and 28 and blood was collected on days 21 and 35. Rabbits were immunized on days 0, 14, 28 and 77 and blood was collected on days 0, 7, 21, 35, 77 and 84. Serum was prepared from the blood samples for serum antibody analyses.

4. Enzyme Linked Immunosorbent Assay (ELISA) of Sera Using In-House Antigen Coated Plates.

[1136] Coating of Plates with Antigen:

[1137] Conjugates 54-BSA and 92-BSA were used as coating antigens. The respective conjugates were diluted to a concentration of 5 μg/mL in phosphate buffered saline (PBS) pH 7.4. 100 μL were coated per well and incubated overnight at 4° C. to get an antigen concentration of 0.5 μg/well. For coating of the isolated PS-II polysaccharide the polysaccharide was diluted to 50 μg/mL in PBS with 10 mM imidazole and 100 μL per well were coated at 50° C. for 5 hours.

Washing:

[1138] After adsorption of the antigen, the plates were washed 1× with PBS-T (200 μL/well) and the excess fluid per well was removed by inverting the plate and tapping on a clean dry tissue towel.

Blocking:

[1139] The plates were blocked using 200 μL of the commercial blocking solution and incubated for 2h at RT.

Washing:

[1140] After blocking, the plates were washed 3× with PBS-T (200 μL/well) and the excess fluid per well was removed by inverting the plate and by tapping on a clean dry tissue towel.

Dilution of Sera and Incubations:

[1141] Pooled sera (n=4 rabbits or n=7-8 mice/group) from different time-points of the different experimental groups were diluted to their respective dilutions in the antibody diluent (PBS+1% BSA). 100 μL of the diluted sera samples of the different experimental groups were added in duplicates to the corresponding wells and incubated on a shaker set at 250 rpm for 2h at RT. For competition ELISA experiments, diluted sera were incubated on ice for 30 min with 10 or 50 μg of isolated PS-II polysaccharide or with PBS before addition to the ELISA plates. 100 μL/well of the antibody diluent (PBS+1% BSA) formed the experimental blank. After incubation with sera, the plates were washed 4× with PBS-T (200 μL/well) and the excess fluid per well was removed by inverting the plate and by tapping on a clean dry tissue towel.

Incubation with Detection Antibody:

[1142] The corresponding detection antibody, anti-rabbit or anti-mouse IgG HRP conjugate was diluted 1:10,000 in the antibody diluent (PBS+1% BSA) and 100 μL/well were added and incubated on a shaker at 250 rpm for 30 min at RT. After the incubation with detection antibody, the plates were washed 5× with PBS-T (200 μL/well) and the excess fluid per well was removed by inverting the plate and by tapping on a clean dry tissue towel.

Substrate Addition:

[1143] To each well, 100 μL of the ready to use TMB (3,3′,5,5′-tetramethylbenzidine) substrate (normalized to RT from 4° C.) was added and incubated in dark for 15 min. The blue color of the enzymatic reaction was stopped by adding 50 μL/well of 2M H.sub.2SO.sub.4 solution resulting in a yellow colored solution. The absorption of the yellow colored solution was measured at 450 nm using a plate reader.

Results:

[1144] The absorption values were analyzed by plotting a graph using the GraphPad Prism software.

5. Enzyme Linked Immunosorbent Assay (ELISA) of Sera Using Commercial Pre-Coated Plates

[1145] This procedure was identical to the above ELISA protocol, except that the coating step was omitted.

Results:

[1146] Serum IgG from immunized rabbits recognizes the immunogen (FIG. 20), the isolated PS-II polysaccharide (FIGS. 19B and 19C) and C. difficile strains 630 (FIGS. 16 and 17), R20291 (FIG. 18) and VPI10463 (FIG. 19A). Serum IgG from immunized mice recognizes the respective immunogens (FIG. 23) and C. difficile strains 630 (FIG. 21) and R20291 (FIG. 22).

[1147] The herein provided data demonstrate that after immunization with a conjugate of the present invention, particularly conjugates 56 and 94, functional antibodies against oligosaccharides of the present invention as well as against the natural C. difficile PS-II polysaccharide, isolated and on the surface of bacteria, were elicited in rabbits and mice. These findings indicate the potential of these antibodies to confer protection infections with C. difficile.

[1148] The ELISA data further proves that the conjugates of the present invention are immunogenic and induce high antibody titers. Hence, ELISA analysis shows that the saccharides of the present invention are immunogenic in rabbits and mice and generate cross-reactive antibodies.