VACCINES AGAINST STREPTOCOCCUS PNEUMONIAE SEROTYPE 4

Abstract

The present invention relates to synthetic saccharides of general formula (I) that are related to Streptococcus pneumoniae serotype 4 capsular polysaccharide and conjugates thereof. Said conjugate and a pharmaceutical composition containing said conjugate are useful for prevention and/or treatment of diseases associated with Streptococcus pneumoniae, and more specifically of diseases associated with Streptococcus pneumoniae serotype 4. Furthermore, the synthetic saccharides of general formula (I) are useful as marker in immunological assays for detection of antibodies against Streptococcus pneumoniae bacteria.

Claims

1. A saccharide of general formula (I)
V*—[U.sub.x+3—U.sub.x+2—U.sub.x+1—U.sub.x].sub.n—V—O-L-NH.sub.2   (I) wherein x is an integer selected from 1, 2, 3 and 4; n is an integer selected from 1, 2 and 3; ##STR00115## —V— represents a bond, —U.sub.x+3—, —U.sub.x+3—U.sub.x+2— or —U.sub.x+3—U.sub.x+2—U.sub.x+1—; V*— represents H—, H—U.sub.x—, H—U.sub.x+1—U.sub.x—, H—U.sub.x+2—U.sub.x+1—U.sub.x—; L represents a linker; or a diastereoisomer or a pharmaceutically acceptable salt thereof.

2. The saccharide according to claim 1 of general formula (II)
V*—[U.sub.x+3—U.sub.x+2—U.sub.x+1—U.sub.x].sub.n—O-L-NH.sub.2   (II) wherein x, n, L, U.sub.X, U.sub.x+1, U.sub.x+2, U.sub.x+3 and V* have the meanings as defined in claim 1.

3. The saccharide according to claim 1 of general formula (III)
V*—[U.sub.x+3—U.sub.x+2—U.sub.x+1—U.sub.x].sub.n—O-L-NH.sub.2   (III) wherein x, n, L, U.sub.X, U.sub.x+1, U.sub.x+2, U.sub.x+3 and V* have the meanings as defined in claim 1.

4. The saccharide according to claim 1 of general formula (IV)
V*—[U.sub.x+3—U.sub.x+2—U.sub.x+1—U.sub.x].sub.n—O-L-NH.sub.2   (IV) wherein x, n, L, U.sub.X, U.sub.x+1, U.sub.x+2, U.sub.x+3 and V* have the meanings as defined in claim 1.

5. The saccharide according to claim 1 of general formula (V)
V*—[U.sub.x+3—U.sub.x+2—U.sub.x+1—U.sub.x].sub.n—O-L-NH.sub.2   (V) wherein x, n, L, U.sub.X, U.sub.x+1, U.sub.x+2, U.sub.x+3 and V* have the meanings as defined in claim 1.

6. The saccharide according to any one of the claims 1-5, wherein x represents 1 and V*— represents H—.

7. A conjugate comprising a saccharide according to any one of the claims 1-6 covalently linked to an immunogenic carrier through the nitrogen atom of the —O-L-NH.sub.2group.

8. A saccharide according to any one of the claims 1-6 or a conjugate according to claim 7 for use in raising a protective immune response in a human and/or animal host.

9. A saccharide according to any one of the claims 1-6 or a conjugate according to claim 7 for use in prevention and/or treatment of diseases associated with bacteria containing in their capsular polysaccharide one of the following saccharide fragments: -3)-β-D-ManNAc-(1,3)-α-L-FucNAc-(1,3)-α-D-GaINAc-(1,4)-α-D-GaI-2,3(S)Pyr-(1-; -4)-α-D-GaI-2,3(S)Pyr-(1,3)-β-D-ManNAc-(1,3)-α-L-FucNAc-(1,3)-α-D-GaINAc-(1-; -3)-α-D-GaINAc-(1,4)-α-D-GaI-2,3(S)Pyr-(1,3)-β-D-ManNAc-(1,3)-α-L-FucNAc-(1-; -3)-α-L-FucNAc-(1,3)-α-D-GaINAc-(1,4)-α-D-GaI-2,3(S)Pyr-(1,3)-β-D-ManNAc-(1-.

10. The saccharide for use or the conjugate for use according to claim 9, wherein the bacteria is Streptococcus pneumoniae serotype 4.

11. The saccharide for use or the conjugate for use according to claim 9 or 10, wherein the diseases associated with bacteria include pneumonia, meningitis, otitis media, bacteremia and acute exacerbation of chronic bronchitis, sinusitis, arthritis and conjunctivitis.

12. A pharmaceutical composition comprising the conjugate according to claim 7 and/or the saccharide according to any one of the claims 1-6 together with at least one pharmaceutically acceptable adjuvant and/or excipient.

13. The pharmaceutical composition according to claim 12 for use in raising a protective immune response in a human and/or animal host.

14. A saccharide according to any one of the claims 1-6 for use as marker in immunological assays for detection of antibodies against bacteria containing in their capsular polysaccharide one of the following saccharide fragments: -3)-β-D-ManNAc-(1,3)-α-L-FucNAc-(1,3)-α-D-GaINAc-(1,4)-α-D-GaI-2,3(S)Pyr-(1-; -4)-α-D-GaI-2,3(S)Pyr-(1,3)-β-D-ManNAc-(1,3)-α-L-FucNAc-(1,3)-α-D-GaINAc-(1-; -3)-α-D-GaINAc-(1,4)-α-D-GaI-2,3(S)Pyr-(1,3)-β-D-ManNAc-(1,3)-α-L-FucNAc-(1-; -3)-α-L-FucNAc-(1,3)-α-D-GaINAc-(1,4)-α-D-GaI-2,3(S)Pyr-(1,3)-β-D-ManNAc-(1-.

Description

DESCRIPTION OF THE FIGURES

[0394] FIG. 1 shows the chemical structure of the S. pneumoniae serotype 4 capsular polysaccharide repeating unit.

[0395] FIG. 2 provides examples of commercially available interconnecting molecules according to the present invention.

[0396] FIG. 3 provides examples of functional group X of the interconnecting molecule according to the present invention.

[0397] FIG. 4 Printing pattern of slides containing pyruvated tetrasaccharide 24* (spot B), depyruvated tetrasaccharide 16* (spot G), trisaccharide 20* (spot I) along with a number of deletion sequences thereof and saccharides with an unnatural stereochemistry thereof. The proteins CRM.sub.197 and the BSA-GlcNAc conjugate were printed to determine antibody response against the carrier protein and the moiety “linker-interconnecting molecule” used during conjugation, respectively. Native S. pneumoniae polysaccharides were printed as controls.

[0398] Spot A: (2S,3aR,4S,6R,7S,7aS)-44(5-aminopentyl)oxy)-7-hydroxy-6-(hydroxyl-methyl)-2-methyltetrahydro-3aH-[1,3]dioxolo[4,5-c]pyran-2-carboxylic acid;

[0399] Spot B: pyruvated tetrasaccharide 24*;

[0400] Spot C: disaccharide 18*;

[0401] Spot D: N-((2S,3R,4R,5R,6R)-2-(((2R,3R,4R,5R,6S)-6-((5-aminopentyl)oxy)-4,5-dihydroxy-2-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)oxy)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)acetamide;

[0402] Spot E: N-((2R,3S,4S,5S,6S)-2-(((2R,3R,4R,5R,6R)-3-acetamido-2-(((2R,3R,4R,5R,6S)-6-((5-aminopentyl)oxy)-4,5-dihydroxy-2-(hydroxyl-methyl)tetrahydro-2H-pyran-3-yl)oxy)-5-hydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-4-yl)oxy)-4,5-dihydroxy-6-methyltetrahydro-2H-pyran-3-yl)acetamide;

[0403] Spot F: S. pneumoniae cell wall polysaccharide (SSI Diagnostica);

[0404] Spot G: tetrasaccharide 16*;

[0405] Spot H: recombinant CRM.sub.197 (Pfenex Inc.);

[0406] Spot I: trisaccharide 20*;

[0407] Spot J: (2R,3S,4S,5S,6R)-2-(((2S,3S,4S,5S,6R)-2-((5-aminopentyl)oxy)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)oxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol;

[0408] Spot L: monosaccharide 21*;

[0409] Spot K: BSA-GlcNAc control conjugate for anti-“linker-interconnecting molecule” antibody response;

[0410] Spot M: S. pneumoniae serotype 4 CPS (SSI Diagnostica);

[0411] Spot N: S. pneumoniae serotype 2 CPS (SSI Diagnostica);

[0412] Spot O: S. pneumoniae serotype 3 CPS (SSI Diagnostica);

[0413] FIG. 5 shows the detection of antibodies against synthetic SP4 based glycans in rabbit SP4 typing serum (SSI Diagnostica) and human reference serum 007sp using glycan array. Competition assay with cell wall polysaccharide (CWPS) and SP4 capsular polysaccharide (CPS) was performed to validate identity of antibodies against SP4 CPS (printing pattern according to FIG. 4). Compared to unpyruvated tetrasaccharide 16*, the signal for the pyruvated saccharide 24* can be inhibited far more efficiently by the native S. pneumoniae serotype 4 CPS suggesting a high number of cross-reactive antibodies. Competition with S. pneumoniae cell wall polysaccharide has no effect on signal strength (see further FIG. 6).

[0414] FIG. 6 shows the quantification of fluorescence intensities for competition experiment with serum 007sp as shown in FIG. 5. Signal decline in competition experiment with serum 007sp is much stronger for pyruvated tetrasaccharide 24* (spot B) compared with unpyruvated tetrasaccharide 16* (spot G) indicating higher antibody cross-reactivity.

[0415] 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.

[0416] 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

[0417] A. Chemical Synthesis

[0418] General Information:

[0419] 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). .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.

Example 1A

Synthesis of (2R,3S,4S,5R,6S)-2-((benzyloxy)methyl)-6-(ethylthio)-4,5-bis(naphthalen-2-ylmethoxy)tetrahydro-2H-pyran-3-ol (1*)

[0420] ##STR00085##

[0421] (2S,4aR,6S,7R,8S,8aS)-6-(ethylthio)-7,8-bis(naphthalen-2-ylmethoxy)-2-phenylhexahydropyrano[3,2-d][1,3]dioxine (S. D. Khaja, V. Kumar, M. Ahmad, J. Xue, K. L. Matta, Tetrahedron Lett. 2010, 51, 4411-4414.) (5.5 g, 9.2 mmol) was stirred in DCM (90 mL) with activated 4 Å MS (5.0 g) for 10 min before cooling to 0° C. Added triethylsilane (11.86 mL, 74.2 mmol) followed by TFA (4.29 mL, 55.7 mmol) dropwise and stirred the reaction mixture at room temperature for 4 h before quenching with water. Extracted the aqueous layer with CH.sub.2Cl.sub.2, and washed the organic layer with sat. aq. NaHCO.sub.3, brine, dried over Na.sub.2SO.sub.4, filtered and concentrated to obtain oil. Purification by flash column chromatography using toluene and acetone as eluent (0 to 7.5%) afforded the compound 1* as colorless oil (4.4 g, 80%). [α].sub.D.sup.20=+29.7° (c=1.10, CHCl.sub.3); IR v.sub.max (film) 3570, 2858, 1362, 1081, 818, 735 cm.sup.−1; .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.93-7.74 (m, 8H), 7.71 (d, J=7.8 Hz, 1H), 7.57 (d, J=8.4 Hz, 1H), 7.52-7.41 (m, 5H), 7.40-7.29 (m, 4H), 5.09 (d, J=9.7 Hz, 1H), 5.01-4.77 (m, 3H), 4.60 (d, J=1.3 Hz, 2H), 4.49 (dd, J=9.7, 1.6 Hz, 1H), 4.17 (s, 1H), 3.88-3.70 (m, 3H), 3.69-3.55 (m, 2H), 2.91-2.66 (m, 2H), 1.35 (td, J=7.4, 1.7 Hz, 3H). .sup.13C NMR (100 MHz, CDCl.sub.3) δ 138.1, 135.9, 135.3, 133.4, 133.3, 133.2 (2C), 128.6, 128.5, 128.2, 128.1 (2C), 127.9 (2C), 127.8 (2C), 127.1, 126.8, 126.5, 126.3, 126.2, 126.1, 126.0, 125.9, 85.3, 82.4, 78.2, 77.1, 76.0, 73.9, 72.3, 69.5, 67.1, 25.0, 15.3; HRMS (ESI): Calcd for C.sub.37H.sub.38O.sub.5S [M+Na].sup.+617.2338, found: 617.2342.

Example 2A

Synthesis of (2R,3S,4S,5R,6S)-2-((benzyloxy)methyl)-6-(ethylthio)-4,5-bis(naphthalen-2-ylmethoxy)tetrahydro-2H-pyran-3-yl benzoate (2*)

[0422] ##STR00086##

[0423] To a 0° C. cooled solution of 1* (4.3 g, 7.23 mmol) in pyridine (30 mL) was added benzoyl chloride (2.52 mL, 21.7 mmol) and stirred at room temperature for 13 h. Diluted the reaction mixture with water and extracted the aqueous layer with ether. Washed the organic layer with water, 1.0 M HCl, brine, dried over Na.sub.2SO.sub.4, filtered and concentrated to obtain oil. Purification by flash column chromatography using hexanes and ethyl acetate as eluent (0 to 10%) afforded the compound 2* as oil (3.8 g, 75%). [α].sub.D.sup.20=+79.9° (c=1.60, CHCl.sub.3); IR v.sub.max (film) 2862, 1721, 1272, 1095, 815, 701 cm.sup.−1; .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.18-8.07 (m, 2H), 7.84-7.71 (m, 6H), 7.69 (d, J=8.7 Hz, 1H), 7.65-7.55 (m, 2H), 7.54-7.34 (m, 9H), 7.32-7.28 (m, 2H), 7.27-7.20 (m, 2H), 5.97 (dd, J=3.0, 0.9 Hz, 1H), 5.04 (t, J=10.9 Hz, 2H), 4.96 (d, J=10.6 Hz, 1H), 4.76 (d, J=11.7 Hz, 1H), 4.59 (d, J=9.2 Hz, 1H), 4.53 (d, J=11.7 Hz, 1H), 4.46 (d, J=11.8 Hz, 1H), 3.91-3.86 (m, 1H), 3.85-3.73 (m, 2H), 3.67 (dd, J=9.5, 5.9 Hz, 1H), 3.60 (dd, J=9.5, 7.0 Hz, 1H), 2.83 (qq, J=12.6, 7.4 Hz, 2H), 1.37 (t, J=7.4 Hz, 3H); .sup.13C NMR (100 MHz, CDCl.sub.3) δ 166.0, 137.7, 135.8, 135.4, 133.8, 133.4 (2C), 133.3, 133.2, 133.1, 130.3, 130.2, 130.0, 128.6 (2C), 128.5, 128.2, 128.1 (2C), 127.9, 127.8 (2C), 127.8, 127.2, 127.0, 126.6, 126.3, 126.1, 125.9, 85.6, 81.3, 78.0, 76.3, 76.1, 73.9, 71.9, 68.5, 67.8, 25.2, 15.3; HRMS (ESI): Calcd for C.sub.44H.sub.42O.sub.6S [M+Na].sup.+721.2600, found: 721.2600.

Example 3A

Synthesis of (2R,3S,4S,5R,6S)-6-((5-(benzyl((benzyloxy)carbonyl)amino)pentyl)oxy)-2-((benzyloxy)methyl)-4,5-bis(naphthalen-2-ylmethoxy)tetrahydro-2H-pyran-3-yl benzoate (3*)

[0424] ##STR00087##

[0425] Stirred a solution of microwave activated 4 Å acid washed molecular sieves (AWMS) (3.2 g), compound 2* (2 g, 2.86 mmol) and C5 aminopentyl linker (1.21 g, 3.72 mmol) in a mixture of ether and DCM (3:1; 36 mL:12 mL) at room temperature for 15 min. Cooled the reaction mixture to 0° C. and added NIS (0.78 g, 3.15 mmol) followed by TfOH (0.25 mL, 0.28 mmol) and stirred for 30 min. Diluted the reaction mixture with aq. sat. Na.sub.2S.sub.2O.sub.3. Extracted the aqueous layer with ether, and dried the organic layer over Na.sub.2SO.sub.4, filtered and concentrated to obtain oil. Purification by flash column chromatography using hexanes and ethyl acetate as eluent (0 to 25%) afforded α-anomer 3* (1.75 g, 63%), and β-anomer (0.63 g, 23%) as oils, indicating a modest selectivity of ˜3:1 (α:β)

[0426] NMR analysis: Because of the C5 aminopentyl linker the anomeric protons in the .sup.1H nmr were submerged or the peaks were broadened and so, the confirmation of the linkage could not be established easily. From .sup.13C for α-anomer, the anomeric carbon was at 98.4 ppm thereby indicating an α-linkage (literature 97 to 101) and the J.sub.C1H1 was 167.7 Hz..sup.x For β-anomer, the .sup.13C value was 104.1 ppm for the anomeric indicating a β-anomer and falls in agreement with literature (103 to 105 ppm)..sup.x Also the J.sub.C1H1 coupling for β-anomer was 158.8 Hz indicating a β-anomer. α-anomer: [α].sub.D.sup.20=+95.8° (c=0.74, CHCl.sub.3); IR v.sub.max (film) 2928, 1720, 1698, 1270, 1103, 1057, 740 cm.sup.−1; .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.00 (dd, J=8.3, 1.2 Hz, 2H), 7.84-7.70 (m, 6H), 7.64 (dd, J=12.7, 5.0 Hz, 2H), 7.56 (t, J=7.4 Hz, 1H), 7.51-7.12 (m, 23H), 5.92 (s, 1H), 5.18 (bs, 2H), 5.03 (d, J=11.4 Hz, 1H), 4.98 (d, J=12.3 Hz, 1H), 4.90 (bs, 1H), 4.84 (d, J=12.2 Hz, 1H), 4.77 (d, J=11.4 Hz, 1H), 4.45 (m, 4H), 4.17 (bs, 2H), 3.99 (dd, J=10.0, 3.6 Hz, 1H), 3.65 (bs, 1H), 3.54 (d, J=6.3 Hz, 2H), 3.44 (bm, 1H), 3.22 (bm, 2H), 1.58 (bm, 4H), 1.31 (bm, 2H); .sup.13C NMR (100 MHz, CDCl.sub.3) δ 171.3, 166.0, 138.1, 137.9, 136.1, 135.9, 133.4, 133.3, 133.1, 133.0, 130.2, 130.0, 128.7, 128.6, 128.5, 128.5, 128.2, 128.1, 128.0 (3C), 127.8 (3C), 127.7, 127.4, 127.0, 126.7, 126.2, 126.1, 126.0, 125.9, 125.8, 98.0, 76.7, 75.2, 73.7, 73.5, 72.1, 69.0, 68.9, 68.4, 68.2, 67.3, 60.5, 50.6, 50.4, 47.3 (2C), 29.3, 28.1, 27.7, 23.6, 21.2, 14.4; HRMS (ESI): Calcd for C.sub.62H.sub.61NO.sub.9 [M+Na].sup.+986.4244, found: 986.4144.

Example 4A

Synthesis of benzyl benzyl(5-(((2S,3R,4S,5S,6R)-6-((benzyloxy)methyl)-5-hydroxy-3,4-bis(naphthalen-2-ylmethoxy)tetrahydro-2H-pyran-2-yl)oxy)pentyl)carbamate (4*)

[0427] ##STR00088##

[0428] To a solution of compound 3* (1.5 g, 1.55 mmol) in a mixture of MeOH and THF (2:1; 10 mL : 5 mL) at room temperature was added a 0.5 M solution of NaOMe in methanol (0.78 mL, 0.39 mmol) and the reaction mixture heated to 50° C. for 30 h. The reaction was neutralized with Amberlite 120 H.sup.+resin, filtered and concentrated. Purification by flash column chromatography using hexanes and ethyl acetate as eluent (0 to 40%) afforded the compound 4* as oil (1.2 g, 90%). [α].sub.D.sup.20=+61.8° (c=2.90, CHCl.sub.3); IR v.sub.max (film) 3462, 2920, 1693, 1226, 1088, 1043, 731 cm.sup.−1; .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.93-7.67 (m, 8H), 7.57-7.41 (m, 6H), 7.39-7.06 (m, 15H), 5.19 (s, 2H), 4.99 (d, J=11.8 Hz, 2H), 4.91 (d, J=11.7 Hz, 1H), 4.84 (bd, J=11.4 Hz, 2H), 4.59 (d, J=12.0 Hz, 1H), 4.55 (d, J=12.0 Hz, 1H), 4.49 (bd, J=12.7 Hz, 2H), 4.15 (s, 1H), 3.96 (d, J=12.3 Hz, 2H), 3.75 (dd, J=10.0, 5.4 Hz, 1H), 3.68 (dd, J=9.9, 6.3 Hz, 1H), 3.63 (d, J=8.3 Hz, 1H), 3.39 (bs, 1H), 3.23 (bm, 2H), 2.69 (s, 1H), 1.75-1.44 (bm, 4H), 1.32 (bm, 2H); .sup.13C NMR (100 MHz, CDCl.sub.3) δ 138.2, 136.1, 135.9, 133.4 (2C), 133.2, 128.7, 128.6, 128.5, 128.4, 128.3, 128.1, 128.0 (2C), 127.8 (3C), 127.4, 126.8, 126.6, 126.3, 126.2, 126.1 (2C), 125.9, 97.5, 77.8, 76.2, 73.7, 73.5, 73.0, 69.8, 68.6, 68.3, 68.2, 67.3, 29.3, 23.6; HRMS (ESI): Calcd for C.sub.55H.sub.57NO.sub.8 [M+Na].sup.+882.3982, found: 882.3918.

Example 5A

Synthesis of (2S,3R,4S,5S,6S)-5-azido-2-methyl-6-(phenylselanyl)tetrahydro-2H-pyran-3,4-diyl diacetate (5*)

[0429] ##STR00089##

[0430] To a solution of (2S,3R,4S)-2-methyl-3,4-dihydro-2H-pyran-3,4-diyl diacetate (14.5 g, 67.7 mmol) and diphenyl diselenide (21.1 g, 67.7 mmol) in CH.sub.2Cl.sub.2 (220 mL) at −50° C. was added bisacetate iodobenzene (21.8 g, 67.7 mmol) followed by trimethylsilyl azide (17.9 mL, 135 mmol). The reaction mixture was warmed to −10° C. over a period of 1.5 h by which time no starting material was observed by TLC. The solvent was removed under vacuum to obtain the crude as reddish brown oil. Purification by flash column chromatography using cyclohexane and ethyl acetate as eluent (0 to 30%) afforded the compound 5* as oil (14.5 g, 52%). [Obtain also other compounds accounting for about 7.6 g whose identity could not be verified using mass and NMR spectroscopy]. IR v.sub.max (film) 2939, 2109, 1742, 1368, 1219, 1083, 1018, 740 cm.sup.−1; .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.63-7.53 (m, 2H), 7.35-7.23 (m, 3H), 5.96 (d, J=5.4 Hz, 1H), 5.33 (dd, J=3.3, 1.3 Hz, 1H), 5.14 (dd, J=10.9, 3.2 Hz, 1H), 4.51 (q, J=6.5, 0.7 Hz, 1H), 4.24 (dd, J=10.9, 5.4 Hz, 1H), 2.18 (s, 3H), 2.07 (s, 3H), 1.10 (d, J=6.5 Hz, 3H); .sup.13C NMR (100 MHz, CDCl.sub.3) δ 170.4, 169.8, 134.8, 129.3, 129.1, 128.3, 128.2, 128.1, 84.5, 71.7, 70.2, 67.6, 58.9, 20.8, 20.7, 15.9; HRMS (ESI): Calcd for C.sub.16H.sub.19N.sub.3O.sub.5Se [M+Na].sup.+436.0388, found: 436.0400.

Example 6A

Synthesis of (2S,3R,4S,5S)-5-azido-2-methyl-6-(2,2,2-trichloro-1-iminoethoxy)tetrahydro-2H-pyran-3,4-diyl diacetate (6*)

[0431] ##STR00090##

[0432] A solution of azidoselenide 5* (5.0 g, 12.1 mmol) in a mixture of THF, water and acetone (1:1:0.5; 28 mL:28 mL:14 mL) was cooled to 0° C. N-iodosuccinimide (5.4 g, 24.2 mmol) was added and the reaction mixture stirred at room temperature for 30 min. The reaction was diluted with ethyl acetate and the organic layer washed with sat. aq. Na.sub.2S.sub.2O.sub.3 and brine respectively. Dried the organic layer over Na.sub.2SO.sub.4, filtered and concentrated to obtain the oil. Purification by flash column chromatography using hexanes and ethyl acetate as eluent (0 to 60%) afforded the compound as a 1:1 mixture of anomers (3.0 g, 91%). Dissolved the lactol (0.75 g, 2.7 mmol) in dichloroethane, and added trichloroacetonitrile (1.37 mL, 13.7 mmol) at room temperature followed by K.sub.2CO.sub.3 (1.02 g, 7.2 mmol) and stirred for 4 h. Filtered the reaction over celite and washed the celite with dichloromethane and removed the solvents under vacuum to obtain the compound 6* as a mixture of anomers (1.12 g, 98%, α:β=1:5.5). The NMR was clean and hence taken to the next step without further purification. [α].sub.D.sup.20=−19.2° (c=1.64, CHCl.sub.3); IR v.sub.max (film) 2993, 2114, 1751, 1729, 1679, 1235, 1216, 1070, 1031, 840, 793 cm.sup.−1; .sup.1H NMR (400 MHz, CDCl.sub.3) reported only β-anomer : δ 8.77 (s, 1H), 5.68 (d, J=8.5 Hz, 1H), 5.25 (dd, J=3.4, 0.9 Hz, 1H), 4.91 (dd, J=10.8, 3.4 Hz, 1H), 3.97-3.83 (m, 2H), 2.21 (s, 3H), 2.08 (s, 3H), 1.24 (d, J=6.4 Hz, 3H); .sup.13C NMR (100 MHz, CDCl.sub.3) δ 170.6, 169.9, 161.1, 97.0, 71.8, 70.5, 69.4, 60.5, 20.8 (2C), 16.2; HRMS (ESI): Calcd for C.sub.12H.sub.15Cl.sub.3N.sub.4O.sub.6 [M+Na].sup.+438.9955, found: 438.9940.

Example 7A

Synthesis of (2S,4aR,6R,7R,8R,8aR)-7-azido-6-(((2R,3S,4S,5R,6S)-6-((5-(benzyl((benzyloxy)carbonyl)amino)pentyl)oxy)-2-((benzyloxy)methyl)-4,5-bis(naphthalen-2-ylmethoxy)tetrahydro-2H-pyran-3-yl)oxy)-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-8-yl acetate (7*)

[0433] ##STR00091##

[0434] To a solution of (2S,4aR,7R,8R,8aR)-7-azido-2-phenyl-6-(2,2,2-trichloro-1-iminoethoxy)hexahydropyrano[3,2-d][1,3]dioxin-8-yl acetate (1.75 g, 2.03 mmol) and acceptor 4* (1.26 g, 2.65 mmol) in a mixture of ether and CH.sub.2Cl.sub.2 (1:1; 11.6 mL: 11.6 mL) at 0° C. was added TMSOTf (0.037 mL, 0.20 mmol) and the reaction mixture stirred at 0° C. for 15 min. Quenched the reaction by adding a drop of Et.sub.3N and removed the solvents under vacuum. Purification by flash column chromatography using hexanes and ethyl acetate as eluent (0 to 20%) afforded α-anomer 7* (1.89 g, 79%) as oil and β-anomer, (0.26 g, 11%) as oil. The selectivity for the glycosylation ranged from 10:1 to 7:1 (03)

[0435] NMR analysis: .sup.1H NMR analysis of α-anomer showed only one anomeric proton that was distinct with J=3.5 Hz. The other anomeric proton was embedded within the napthyl methylene protons. For the β-anomer the coupling constant was J=8.1 Hz. .sup.13C indicated a value of 99.5 and 97.1 ppm for α-anomer and 101.8 and 97.7 ppm for β-anomer. The J.sub.C1H1 coupling for fraction-1 was 170.4 Hz and 166.2 Hz indicating two α-anomeric linkages and was 168.6 Hz and 164.3 Hz for β-anomer indicating one α- and one β-anomeric linkages. α-anomer: [α].sub.D.sup.20=+129.0° (c=1.25, CHCl.sub.3); IR v.sub.max (film) 2925, 2109, 1744, 1694, 1225, 1089, 1042, 748 cm.sup.−1; .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.90 (s, 1H), 7.87-7.70 (m, 7H), 7.58 (d, J=8.6 Hz, 1H), 7.53-7.04 (m, 25H), 5.24 (dd, J=11.1, 3.3 Hz, 1H), 5.19 (s, 1H), 5.17 (s, 2H), 5.08 (d, J=3.5 Hz, 1H), 4.97 (d, J=11.9 Hz, 1H), 4.94-4.81 (m, 4H), 4.54 (s, 2H), 4.47 (d, J=10.0 Hz, 2H), 4.30 (d, J=2.7 Hz, 1H), 4.14 (d, J=2.9 Hz, 1H), 4.07 (s, 1H), 4.02 (dd, J=10.3, 3.6 Hz, 1H), 3.98-3.86 (m, 4H), 3.64-3.46 (m, 3H), 3.35 (bs, 1H), 3.20 (bm, 2H), 3.02 (d, J=11.7 Hz, 1H), 2.16 (s, 3H), 1.56 (bm, 4H), 1.38-1.06 (bm, 2H); .sup.13C NMR (100 MHz, CDCl.sub.3) δ 170.5, 138.1, 137.8, 137.7, 136.1, 133.4 (2C), 133.2, 133.0, 129.1, 128.7, 128.6 (2C), 128.3 (2C), 128.2 (2C), 128.1, 128.0 (2C), 127.9, 127.8, 127.4, 127.2, 126.6, 126.3 (2C), 126.2, 126.0 (2C), 125.9, 125.5, 100.5, 99.0, 97.3, 77.2, 76.5, 74.6, 73.7, 73.4, 73.3, 73.1, 70.4, 69.0, 68.8, 68.3, 67.2, 62.3, 58.0, 29.3, 23.6, 21.2; HRMS (ESI): Calcd for C.sub.70H.sub.72N.sub.4O.sub.13 [M+Na].sup.+1199.4994, found: 1199.4902.

Example 8A

Synthesis of benzyl (5-(((2S,3R,4S,5S,6R)-5-(((2S,4aR,6R,7R,8R,8aR)-7-azido-8-hydroxy-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-6-yl)oxy)-6-((benzyloxy)methyl)-3,4-bis(naphthalen-2-ylmethoxy)tetrahydro-2H-pyran-2-yl)oxy)pentyl)(benzyl)carbamate (8*)

[0436] ##STR00092##

[0437] To a solution of the compound 7* (1.85 g, 1.57 mmol) in a mixture of methanol and THF (2:1; 16 mL : 8 mL) was added a 0.5 M solution of NaOMe in MeOH (0.31 mL, 0.15 mmol) and stirred for 12 h at room temperature. The reaction was neutralized using Amberlite 120 H.sup.+resin, filtered, and concentrated. Purification by flash column chromatography using hexanes and ethyl acetate as eluent (0 to 20%) afforded the compound 8* (1.74 g, 98%) as white foam. [α].sub.D.sup.20=+88.9° (c=1.15, CHCl.sub.3); IR v.sub.max (film) 3474, 2925, 2111, 1740, 1694, 1234, 1088, 1041, 748 cm.sup.−1; .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.92-7.68 (m, 8H), 7.58 (d, J=8.2 Hz, 1H), 7.55-7.43 (m, 5H), 7.42-7.07 (m, 20H), 5.18 (bs, 3H), 5.01 (d, J=3.4 Hz, 1H), 4.98 (s, 1H), 4.94 (s, 1H), 4.92-4.78 (m, 3H), 4.54 (s, 2H), 4.48 (bd, J=9.2 Hz, 2H), 4.28 (s, 1H), 4.03-3.83 (m, 6H), 3.66-3.52 (m, 4H), 3.50 (dd, J=10.5, 3.4 Hz, 1H), 3.47-3.32 (bm, 1H), 3.30-3.13 (m, 2H), 3.07 (d, J=11.6 Hz, 1H), 1.79-1.43 (bm, 4H), 1.41-1.10 (bm, 2H); .sup.13C NMR (100 MHz, CDCl.sub.3) δ 138.1, 137.7, 137.6, 136.1, 135.9, 133.4, 133.3, 133.2, 133.0, 129.4, 128.7, 128.6 (2C), 128.4, 128.3, 128.2 (2C), 128.1 (2C), 128.0, 127.9 (2C), 127.3, 126.7, 126.4 (2C), 126.3, 126.2, 126.1, 125.9, 125.6, 101.0, 99.2, 97.0, 77.2, 75.5, 75.4, 74.5, 73.7, 73.0, 72.8, 69.1, 69.0, 68.3, 67.7, 67.3, 62.6, 61.4, 29.3, 23.6; HRMS (ESI): Calcd for C.sub.68H.sub.70N.sub.4O.sub.12 [M+Na].sup.+1157.4888, found: 1157.4923.

Example 9A

Synthesis of benzyl (5-(((2S,3R,4S,5S,6R)-5-(((2S,4aR,6R,7R,8R,8aR)-7-azido-8-(((2S,3S,4S,5S,6S)-3-azido-4,5-dihydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-6-yl)oxy)-6-((benzyloxy)methyl)-3,4-bis(naphthalen-2-ylmethoxy)tetrahydro-2H-pyran-2-yl)oxy)pentyl)(benzyl)carbamate (9*)

[0438] ##STR00093##

[0439] To a solution of donor compound (2S,3R,4S,5S)-5-azido-2-methyl-6-(2,2,2-trichloro-1-iminoethoxy)tetrahydro-2H-pyran-3,4-diyl diacetate 6* (0.5 g, 1.2 mmol) and acceptor 8* (1.05 g, 0.92 mmol) in DCM (10 mL) at −20° C. was added TMSOTf (0.017 mL, 0.092 mmol) and the reaction mixture warmed to 0° C. over 30 min. Quenched the reaction by addition of two drops of Et.sub.3N, and evaporated. Purification by flash column chromatography using toluene and acetone as eluent (0 to 25%) afforded the intermediate trisaccharide as a mixture of anomers that could not be separated easily at this step. To a solution of intermediate trisaccharide in a mixture of methanol and THF (2:1; 7 mL : 3.5 mL) was added 0.5 M solution of NaOMe in MeOH (0.185 mL, 0.092 mmol) and stirred at room temperature for 12 h. The reaction was neutralized with Amberlite 120 H.sup.+ resin, filtered, and concentrated. Purification by flash column chromatography using hexanes and ethyl acetate as eluent (0 to 25%) afforded α-anomer 9* (0.70 g, 58%) and β-anomer (0.12 g, 14%) as white foams.

[0440] NMR analysis: .sup.1H NMR of α-anomer contained three α-anomeric protons with chemical shift and coupling constant of 5.09 ppm (J=3.5 Hz), 4.99 ppm, and 4.83 ppm (J=3.6 Hz). .sup.13C had values of 100.5, 99.2, and 97.0 ppm. The J.sub.C1H1 coupling was 174.5, 168.4, and 167.0 Hz indicating three α-anomeric linkages. .sup.1H nmr of (β-anomer contained two α-anomeric protons based on chemical shift and coupling constant of 5.00 ppm (J=3.5 Hz), and 4.91 ppm and he β-anomeric proton at 4.06 ppm (J=7.1 Hz). .sup.13C had a value of 99.1 (2C) and 97.0 ppm The J.sub.C1H1 coupling was 168.6, and 167.1 Hz indicating two α-anomeric linkages, the β linkage overlapped with one of the α and hence could not be calculated. [α].sub.D.sup.20=+71.6° (c=1.06, CHCl.sub.3); IR v.sub.max (film) 3488, 2923, 2111, 1740, 1694, 1234, 1088, 1040, 747 cm.sup.−1; .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.89 (s, 1H), 7.87-7.78 (m, 5H), 7.78-7.71 (m, 2H), 7.57 (d, J=8.2 Hz, 1H), 7.52-7.42 (m, 5H), 7.41-7.08 (m, 20H), 5.21 (s, 1H), 5.18 (bs, 2H), 5.09 (d, J=3.5 Hz, 1H), 5.00 (bs, 1H), 4.95 (bs, 2H), 4.91 (bs, 2H), 4.83 (d, J=3.7 Hz, 1H), 4.60-4.50 (m, 2H), 4.47 (bs, 2H), 4.34 (s, 1H), 4.17 (q, J=7.4 Hz, 1H), 4.22-4.11 (m, 1H), 4.10-3.85 (m, 6H), 3.83 (dd, J=10.8, 3.2 Hz, 1H), 3.72 (dd, J=10.8, 3.5 Hz, 1H), 3.70-3.50 (m, 5H), 3.41 (bs, 1H), 3.21 (bm, 2H), 3.09 (d, J=11.7 Hz, 1H), 2.47 (s, 1H, OH), 2.23 (s, 1H, OH), 1.59 (bm, 4H), 1.28 (bm, 2H), 1.14 (d, J=6.6 Hz, 3H); .sup.13C NMR (100 MHz, CDCl.sub.3) δ 138.1, 137.8, 137.7, 136.1, 136.0, 133.4 (2C), 133.2, 133.1, 129.2, 128.7 (2C), 128.7, 128.6, 128.4, 128.2, 128.1, 128.0 (2C), 127.9, 127.1, 126.4 (2C), 126.3, 126.1 (2C), 125.9, 125.6, 100.9, 100.5, 99.3, 97.0, 77.5, 77.4 (2C), 77.2, 77.0, 76.9, 75.7, 75.5, 74.1, 73.7, 73.1, 72.6, 71.7, 69.2, 69.0, 68.8, 68.3, 67.4, 67.3, 66.7, 62.4, 61.1, 59.0, 29.3, 23.6, 16.5; HRMS (ESI): Calcd for C.sub.74H.sub.79N.sub.7O.sub.15 [M+Na].sup.+1328.5532, found: 1328.5551.

Example 10A

Synthesis of (2S,3S,4S,5S,6S)-5-azido-6-(((2S,4aR,6R,7R,8R,8aR)-7-azido-6-(((2R,3S,4S,5R,6S)-6-((5-(benzyl((benzyloxy)carbonyl)amino)pentyl)oxy)-2-((benzyloxy)methyl)-4,5-bis(naphthalen-2-ylmethoxy)tetrahydro-2H-pyran-3-yl)oxy)-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-8-yl)oxy)-4-hydroxy-2-methyltetrahydro-2H-pyran-3-yl acetate (10*)

[0441] ##STR00094##

[0442] To a solution of the trisaccharide compound 9* (0.65 g, 0.40 mmol) in DMF (2.4 mL) at room temperature was added trimethyl orthoacetate (0.38 mL, 2.99 mmol) and p-TSA (0.014 g, 0.075 mmol) and the reaction mixture stirred for 30 min. Triethylamine (4 drops) was added and the solvent removed under vacuum using toluene as an azeotrop. To the crude was added 80% acetic acid (4.66 mL) and the reaction mixture stirred for 1 h at room temperature. The solvent was removed under vacuum, azeotroped with toluene to obtain oil. Purification by flash column chromatography using hexanes and ethyl acetate as eluent (10 to 50%) afforded the compound 10* as white foam (0.62 g, 92%). [α].sub.D.sup.20=+66.5° (c=1.10, CHCl.sub.3); IR v.sub.max (film) 2925, 2112, 1746, 1697, 1233, 1089, 1042, 746 cm.sup.−1; .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.90 (s, 1H), 7.89-7.79 (m, 5H), 7.76 (dd, J=6.7, 2.6 Hz, 2H), 7.58 (d, J=8.2 Hz, 1H), 7.54-7.43 (m, 5H), 7.42-7.21 (m, 19H), 7.17 (s, 1H), 5.20 (bs, 3H), 5.10 (d, J=3.5 Hz, 1H), 5.08 (dd, J=3.4, 1.2 Hz, 1H), 5.01 (bs, 1H), 4.98-4.89 (m, 4H), 4.83 (d, J=3.7 Hz, 1H), 4.61-4.51 (m, 2H), 4.48 (d, J=6.7 Hz, 2H), 4.35 (s, 1H), 4.26 (q, J=6.4 Hz, 1 H), 4.19 (dd, J=10.7, 3.5 Hz, 1H), 4.05 (d, J=3.0 Hz, 1H), 4.04-3.93 (m, 4H), 3.89 (t, J=8.8 Hz, 1H), 3.83 (dd, J=10.8, 3.2 Hz, 1H), 3.72 (dd, J=10.8, 3.4 Hz, 1H), 3.67 (d, J=11.7 Hz, 1H), 3.64-3.50 (m, 3H), 3.42 (bs, 1H), 3.22 (bm, 2H), 3.10 (d, J=11.7 Hz, 1H), 2.22 (s, 3H), 1.57 (bm, 4H), 1.40-1.16 (bm, 2H), 1.00 (d, J=6.6 Hz, 3H).; .sup.13C NMR (100 MHz, CDCl.sub.3) δ 171.5, 138.1, 137.7, 136.0 (2C), 133.4 (2C), 133.2, 133.1, 129.3, 128.7, 128.6 (2C), 128.4 (2C), 128.2, 128.1, 128.0 (3C), 127.9, 127.4, 127.1, 126.4, 126.3, 126.1 (2C), 125.9, 125.6, 101.0, 100.5, 99.2, 97.0, 77.4, 77.1, 75.7, 75.5, 74.0, 73.7, 73.1, 72.5, 69.2, 68.9, 68.3, 67.6, 67.3 (2C), 65.8, 62.3, 61.0, 59.0, 29.3, 23.6, 21.0, 16.5.; HRMS (ESI): Calcd for C.sub.76H.sub.81N.sub.7O.sub.16 [M+Na].sup.+1370.5637, found: 1370.5479.

Example 11A

Synthesis of (2R,4aR,6R,7R,8S,8aR)-6-(((2S,3R,4S,5S,6S)-3-acetoxy-5-azido-6-(((2S,4aR,6R,7R,8R,8aR)-7-azido-6-(((2R,3S,4S,5R,6S)-6-((5- (benzyl((benzyloxy)carbonyl)amino)pentyl)oxy)-2-((benzyloxy)methyl)-4,5-bis(naphthalen-2-ylmethoxy)tetrahydro-2H-pyran-3-yl)oxy)-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-8-yl)oxy)-2-methyltetrahydro-2H-pyran-4-yl)oxy)-8-(benzyloxy)-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-7-yl 4-oxopentanoate (11*)

[0443] ##STR00095##

[0444] Stirred a solution of acceptor 10* (0.25 g, 0.18 mmol), (2R,4aR,6S,7R,8S,8aR)-8-(benzyloxy)-6-(ethylthio)-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-7-yl 4-oxopentanoate (0.14 g, 0.27 mmol) (dried under vacuum overnight) and activated 4 Å MS (0.39 g) in DCM (2 mL) for 1 h at room temperature. After cooling to −30° C. added NIS (0.063 g, 0.27 mmol) followed by TfOH (8.2 μL, 0.093 mmol) and stirred the reaction mixture for 1 h. Quenched the reaction using 0.05 mL of Et.sub.3N. Diluted the RM with DCM and washed the organic layer with sat. aq. Na.sub.2S.sub.2O.sub.3, water, brine, dried over Na.sub.2SO.sub.4, filtered and concentrated to obtain yellow oil. Purified the crude by flash chromatography using hexanes and ethyl acetate as eluent (10% to 50%) to obtain the compound 11* as white foam (0.22, 66%).

[0445] NMR analysis: .sup.1H NMR indicated three α- and one β-anomeric protons based on chemical shift and coupling constant at 5.02 ppm (J=3.5 Hz), 4.98 ppm, and 4.70 ppm (J=3.6 Hz) for α and 4.57 ppm (J=7.6 Hz) for β. .sup.13C indicated a value of 100.8, 99.1 and 97.0 ppm for α and 100.0 for β. The J.sub.C1H1 coupling was 173.9, 172.0, and 170.6 Hz for three α-anomeric and 164.9 Hz for the β-anomeric linkages. [α].sub.D.sup.20=+36.6° (c=0.90, CHCl.sub.3); IR v.sub.max (film) 2925, 2111, 1746, 1696, 1233, 1089, 1042, 748 cm.sup.−1; .sup.1H NMR (400 MHz, Acetone-d.sub.6) δ 8.02-7.73 (m, 8H), 7.64 (d, J=8.1 Hz, 1H), 7.60 (dd, J=8.5, 1.6 Hz, 1H), 7.55-7.20 (m, 34H), 5.71 (s, 1H), 5.36 (s, 1H), 5.25 (d, J=2.6 Hz, 1H), 5.15 (m, 3H), 5.06 (s, 1H), 5.02-4.86 (m, 6H), 4.83 (d, J=6.7 Hz, 1H), 4.80 (d, J=2.6 Hz, 1H), 4.72 (d, J=11.9 Hz, 1H), 4.60 (s, 2H), 4.51 (s, 2H), 4.39 (s, 1H), 4.34 (dd, J=10.9, 3.3 Hz, 1H), 4.30-4.20 (m, 3H), 4.17 (s, 1H), 4.09-3.91 (m, 5H), 3.86-3.70 (m, 4H), 3.71-3.59 (m, 3H), 3.57 (dd, J=10.9, 3.7 Hz, 1H), 3.54-3.38 (m, 2H), 3.33 (dd, J=12.4, 1.4 Hz, 1H), 3.24 (bs, 2H), 2.78-2.64 (m. 2H), 2.64-2.48 (m, 2H), 2.13 (s, 3H), 2.12 (s, 3H), 1.57 (bs, 4H), 1.37 (bs, 2H), 0.98 (d, J=6.5 Hz, 3H); .sup.13C NMR (100 MHz, Acetone-d.sub.6) δ 172.0, 170.9, 139.7 (2C), 139.6, 139.4, 139.0, 137.7, 134.4, 134.3, 134.0, 133.9, 129.8, 129.7, 129.6, 129.3 (2C), 129.2, 129.1, 129.0, 128.9 (2C), 128.8 (2C), 128.7 (2C), 128.6 (2C), 128.5, 128.3, 128.0, 127.5, 127.3, 127.2 (2C), 127.1, 127.0, 126.8, 126.7, 126.5, 126.2, 101.8, 101.6, 101.4, 100.1, 98.0, 82.1, 80.2, 78.3, 78.3, 76.4, 76.3, 75.2, 74.6, 74.2, 74.1, 73.9, 73.1, 72.6, 71.1, 70.0, 69.6, 69.3, 68.6, 67.5, 66.8, 66.7, 63.4, 60.3, 59.7, 38.2, 30.0, 29.9, 28.7, 24.2, 20.8, 16.8; HRMS (ESI): Calcd for C.sub.101H.sub.107N.sub.7O.sub.23 [M+Na].sup.+1808.7316, found: 1808.7196.

Example 12A

Synthesis of (2S,3R,4S,5S,6S)-5-azido-6-(((2S,4aR,6R,7R,8R,8aR)-7-azido-6-(((2R,3S,4S,5R,6S)-6-((5-(benzyl((benzyloxy)carbonyl)amino)pentyl)oxy)-2-((benzyloxy)methyl)-4,5-bis(naphthalen-2-ylmethoxy)tetrahydro-2H-pyran-3-yl)oxy)-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-8-yl)oxy)-4-(((2R,4aR,6R,7R,8R,8aR)-8-(benzyloxy)-7-hydroxy-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-6-yl)oxy)-2-methyltetrahydro-2H-pyran-3-yl acetate (12*)

[0446] ##STR00096##

[0447] Compound 11* (0.18 g, 0.10 mmol) was dissolved in a mixture of toluene, ethanol and DCM (2:1:0.5; 3.6 mL : 1.8 mL : 0.9 mL). Hydrazine acetate (0.046 g, 0.50 mmol) was then added. After 30 min at room temperature diluted the reaction with water and extracted the aqueous with ether. Washed the organic layer with brine, dried over Na.sub.2SO.sub.4, filtered, and concentrated to obtain oil. Purified the crude by flash chromatography using hexanes and ethyl acetate as eluent, (0 to 30%) to obtain the compound 12* as oil (0.12 g, 70%). [α].sub.D.sup.20 =+42.0° (c=1.00, CHC.sub.3); IR v.sub.max (film) 3494, 2869, 2111, 1730, 1695, 1234, 1088, 1041, 746 cm.sup.−1; .sup.1H NMR (400 MHz, Acetone-d.sub.6) δ 8.04-7.73 (m, 8H), 7.64 (d, J=8.0 Hz, 1H), 7.60 (dd, J=8.5, 1.6 Hz, 1H), 7.56-7.07 (m, 34H), 5.66 (s, 1H), 5.38 (s, 1H), 5.34 (d, J=2.5 Hz, 1H), 5.16 (d, J=3.1 Hz, 3H), 5.06 (s, 1H), 5.02-4.88 (m, 5H), 4.86 (q, J=12.0 Hz, 2H), 4.62 (s, 1H), 4.61 (s, 2H), 4.51 (s, 2H), 4.41 (s, 1H), 4.38 (dd, J=10.9, 3.3 Hz, 1H), 4.33 (q, J=6.1 Hz, 1H), 4.28 (d, J=2.7 Hz, 1H), 4.24-4.17 (m, 2H), 4.14 (d, J=4.0 Hz, 1H), 4.10-3.93 (m, 5H), 3.83-3.71 (m, 2H), 3.70-3.55 (m, 6H), 3.49-3.38 (m, 3H), 3.34 (dd, J=12.4, 1.5 Hz, 1H), 3.24 (bs, 2H), 2.17 (s, 3H), 1.57 (bs, 4H), 1.42-1.24 (bs, 2H), 1.03 (d, J=6.5 Hz, 3H); .sup.13C NMR (100 MHz, Acetone-d.sub.6) δ 171.1, 139.5, 138.8, 138.7, 138.5, 138.3, 136.8 (2C), 133.5, 133.4, 133.1, 133.0, 128.7, 128.6, 128.4 (2C), 128.3, 128.0 (2C), 127.9 (3C), 127.8, 127.7 (2C), 127.6, 127.5, 127.1, 126.6, 126.4, 126.3, 126.2 (2C), 126.1, 125.9, 125.8, 125.6, 101.5, 101.0, 100.7, 100.5, 99.2, 97.1, 81.3, 81.1, 77.4, 77.2, 75.6, 75.4, 74.7, 74.6, 74.2, 74.0, 73.5, 73.0, 72.3, 71.7, 70.2, 69.1, 68.7, 68.5, 67.7, 66.6, 66.2, 65.7, 62.6, 59.2, 59.02, 29.1, 23.3, 20.1, 15.9; HRMS (ESI): Calcd for C.sub.96H.sub.101N.sub.7O.sub.21 [M+Na].sup.+1710.6948, found: 1710.6809.

Example 13A

Synthesis of (2S,3R,4S,5S,6S)-5-azido-6-(((2S,4aR,6R,7R,8R,8aR)-7-azido-6-(((2R,3S,4S,5R,6S)-64(5-(benzyl((benzyloxy)carbonyl)amino)pentyl)oxy)-2-((benzyloxy)methyl)-4,5-bis(naphthalen-2-ylmethoxy)tetrahydro-2H-pyran-3-yl)oxy)-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-8-yl)oxy)-4-(((2R,4aR,6R,7S,8R,8aS)-7-azido-8-(benzyloxy)-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-6-yl)oxy)-2-methyltetrahydro-2H-pyran-3-yl acetate (13*)

[0448] ##STR00097##

[0449] To a solution of compound 12* (0.05 g, 0.03 mmol) in DCM (0.6 mL) was added pyridine (0.06 mL, 0.74 mmol) followed by a 1.0 M solution of triflic anhydride in DCM (0.089 mL, 0.089 mmol) and stirred the reaction mixture at room temperature for 30 min. Quenched the reaction with sat. aq. NaHCO.sub.3 and extracted the aqueous with DCM, washed with brine, dried over Na.sub.2SO.sub.4, filtered, and concentrated to obtain oil which was dried on high vacuum for 2 h. The crude was then taken in DMF (0.6 mL), added NaN.sub.3 (0.0057 g, 0.089 mmol) and heated the reaction to 80° C. for 1.5 h. After cooling to room temperature, diluted the reaction with sat. aq. NH.sub.4C1 and extracted the aqueous layer with ethyl acetate. Washed the organic layer with brine, dried over Na.sub.2SO.sub.4, filtered, and concentrated to obtain oil. Purified the crude by flash chromatography using hexanes and ethyl acetate as eluent (0 to 40%) to obtain the compound 13* as white foam (0.031 g, 61%).

[0450] NMR analysis: .sup.1H NMR showed a coupling constant of J=1.1 Hz for the β-mannoside linkage which before inversion was 7.2 Hz.

[0451] [α].sub.D.sup.20=+21.0° (c=1.50, CHCl.sub.3); IR v.sub.max (film) 2916, 2860, 2112, 1737, 1697, 1234, 1088, 1045, 746 cm.sup.−1; .sup.1H NMR (400 MHz, Acetone-d.sub.6) δ 8.01-7.76 (m, 8H), 7.64 (d, J=8.6 Hz, 1H), 7.60 (dd, J=8.5, 1.5 Hz, 1H), 7.55-7.17 (m, 34H), 5.68 (s, 1H), 5.36 (s, 1H), 5.30 (d, J=3.3 Hz, 1H), 5.15 (d, J=3.4 Hz, 3H), 5.07 (s, 1H), 5.02 (d, J=1.3 Hz, 1H), 5.01-4.90 (m, 5H), 4.82 (d, J=12.2 Hz, 1H), 4.74 (d, J=12.2 Hz, 1H), 4.60 (s, 2H), 4.51 (s, 2H), 4.40 (s, 1H), 4.37 (dd, J=10.9, 3.3 Hz, 1H), 4.35-4.23(m, 2H), 4.17 (s, 1H), 4.14 (dd, J=10.4, 4.9 Hz, 1H), 4.09-3.94 (m, 6H), 3.93-3.83 (m, 2H), 3.79 (t, J=10.3 Hz, 1H), 3.74 (dd, J=10.9, 3.4 Hz, 1H), 3.63 (m, 3H), 3.55 (dd, J=10.9, 3.7 Hz, 1H), 3.51-3.36 (m, 2H), 3.33 (d, J=11.0 Hz, 1H), 3.24 (bs, 2H), 2.16 (s, 3H), 1.57 (bs, 4H), 1.36 (bs, 2H), 1.04 (d, J=6.5 Hz, 3H).; .sup.13C NMR (100 MHz, Acetone-d.sub.6) δ 171.5, 139.7, 139.6 (2C), 139.4, 139.1, 137.7 (2C), 134.4, 134.3, 134.0, 133.9, 129.7, 129.6, 129.3 (2C), 129.2, 129.1, 128.9 (3C), 128.8, 128.7, 128.6 (2C), 128.5, 128.4, 128.0, 127.5, 127.3, 127.2, 127.1, 127.0, 126.8, 126.7, 126.5, 102.2, 101.5 (2C), 100.2, 98.7, 98.0, 79.2, 78.3, 78.2, 77.7, 76.5, 76.3, 75.1, 74.3, 73.9, 73.2, 73.1, 72.6, 70.8, 69.9, 69.6, 69.1, 68.6, 68.0, 67.5, 66.2, 64.7, 63.4, 60.1, 59.7, 30.0, 24.2, 20.9, 16.7; HRMS (ESI): Calcd for C.sub.96H.sub.100N.sub.10O.sub.20 [M+Na].sup.+1735.7013, found: 1735.6995.

Example 14A:

Synthesis of (2S,3R,4S,5S,6S)-5-acetamido-6-(((2S,4aR,6R,7R,8R,8aR)-7-acetamido-6-(((2R,3S,4S,5R,6S)-6-((5-(benzyl((benzyloxy)carbonyl)amino)pentyl)oxy)-2-((benzyloxy)methyl)-4,5-bis(naphthalen-2-ylmethoxy)tetrahydro-2H-pyran-3-yl)oxy)-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-8-yl)oxy)-4-(((2R,4aR,6R,7S,8R,8aS)-7-acetamido-8-(benzyloxy)-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-6-yl)oxy)-2-methyltetrahydro-2H-pyran-3-yl acetate (14*)

[0452] ##STR00098##

[0453] To a solution of compound 13* (0.03 g, 0.018 mmol) in pyridine (0.5 mL) was added thioacetic acid (0.15 mL, 2.18 mmol) and the reaction stirred at room temperature for 96 h. (Since the reaction was not able to be monitored by TLC, LC-MS was taken after 48 h and it showed the presence of diacetamide. Added further 0.07 mL of thioacetic acid and continued stirring for additional 48 h (LC-MS showed no starting material or mono- or diacetamide). The solvent were removed under vacuo and the crude azeotroped twice with toluene. Purified the crude by flash chromatography using DCM, acetone and MeOH as eluent (5% each of MeOH and acetone upto 50%) to obtain the compound 14* as foam (0.025 g, 81%). .sub.[1:1]D20=+28.0° (c=1.38, CHCl.sub.3); IR v.sub.max (film) 3434, 2934, 2860, 1738, 1674, 1234, 1093, 1045, 749 cm.sup.−1; .sup.1H NMR (400 MHz, Acetone-d.sub.6) δ 7.89 (m, 8H), 7.74 (d, J=7.9 Hz, 1H), 7.65-7.54 (m, 2H), 7.55-7.14 (m, 34H), 6.47 (d, J=9.8 Hz, 1H), 6.24 (d, J=9.1 Hz, 1H), 5.55 (s, 1H), 5.44 (s, 1H), 5.24 (d, J=2.5 Hz, 1H), 5.14 (s, 2H), 5.10 (d, J=3.6 Hz, 1H), 5.06-4.94 (m, 5H), 4.90 (d, J=12.7 Hz, 1H), 4.81 (d, J=1.7 Hz, 1H), 4.77 (d, J=11.9 Hz, 1H), 4.74-4.66 (m, 1H), 4.57 (d, J=11.7 Hz, 1H), 4.53-4.45 (m, 5H), 4.41 (s, 1H), 4.39-4.28 (m, 3H), 4.28-4.22 (m, 1H), 4.20 (dd, J=10.0, 4.7 Hz, 1H), 4.11 (dd, J=10.3, 3.4 Hz, 1H), 4.08-3.97 (m, 4H), 3.84 (t, J=9.6 Hz, 1H), 3.79-3.52 (m, 6H), 3.40 (m, 3H), 3.22 (s, 2H), 2.13 (s, 3H), 1.96 (s, 3H), 1.94 (s, 3H), 1.94 (s, 3H), 1.56 (bs, 4H), 1.41-1.30 (m, 2H), 1.14 (d, J=6.5 Hz, 3H).; .sup.13C NMR (100 MHz, Acetone-d.sub.6) δ 171.6, 171.0, 170.4 (2C), 139.8 (2C), 139.5, 139.3, 139.2, 137.8, 137.6, 134.3 (2C), 134.0, 133.9, 129.5, 129.3, 129.2, 129.2, 128.9, 128.8 (4C), 128.7, 128.6 (4C), 128.5 (2C), 128.0 (2C), 127.2, 127.1, 127.0, 126.9, 126.8, 126.6, 126.4 (2C), 102.2, 101.9, 101.4, 100.0, 98.4, 98.2, 79.1, 78.6, 77.3, 76.9, 76.7 (2C), 73.8, 73.7, 73.0 (2C), 71.3, 70.5, 70.2, 69.8, 69.2, 68.7 (2C), 68.0, 67.4, 65.8, 63.3, 50.8, 49.6, 49.4, 30.0, 24.2, 23.8, 23.7, 23.2, 20.8, 16.9; HRMS (ESI): Calcd for C.sub.102H.sub.112N.sub.4O.sub.23 [M+Na].sup.+1783.7615, found: 1783.7609.

Example 15A

Synthesis of benzyl (5-(((2S,3R,4S,5S,6R)-5-(((2S,4aR,6R,7R,8R,8aR)-7-acetamido-8-(((2S,3S,4S,5R,6S)-3-acetamido-4-(((2R,4aR,6R,7S,8R,8aS)-7-acetamido-8-(benzyloxy)-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-6-yl)oxy)-5-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-6-yl)oxy)-6-((benzyloxy)methyl)-3,4-bis(naphthalen-2-ylmethoxy)tetrahydro-2H-pyran-2-yl)oxy)pentyl)(benzyl)carbamate (15*)

[0454] ##STR00099##

[0455] To a solution of compound 14*(0.021 g, 0.012 mmol) in MeOH (0.45 mL) was added a solution of 0.5 M NaOMe in MeOH (5.96 μL, 2.98 μmol) and the reaction stirred for 2.5 h at room temperature. Diluted the reaction with MeOH, neutralized with

[0456] Amberlite 120 H.sup.+resin, filtered, and concentrated to obtain the compound 15* as foam (0.0193 g, 94%). [α].sub.D.sup.20=+49.1° (c=1.90, CHCl.sub.3); IR v.sub.max (film) 3354, 2929, 2860, 1667, 1372, 1096, 1045, 751 cm.sup.−1; .sup.1H NMR (400 MHz, Acetone-d.sub.6) δ 8.02-7.78 (m, 8H), 7.73 (d, J=7.9 Hz, 1H), 7.63-7.53 (m, 2H), 7.53-7.17 (m, 34H), 7.10 (d, J=9.5 Hz, 1H), 6.46 (d, J=9.1 Hz, 1H), 5.56 (s, 1H), 5.42 (s, 1H), 5.14 (s, 2H), 5.10 (d, J=3.6 Hz, 1H), 5.05-4.81 (m, 9H), 4.78-4.68 (m, 1H), 4.61-4.45 (m, 6H), 4.45-4.25 (m, 4H), 4.20 (dd, J=10.1, 4.8 Hz, 1H), 4.17 (d, J=6.5 Hz, 1H), 4.13 (dd, J=10.3, 3.6 Hz, 1H), 4.09-3.97 (m, 3H), 3.94 (d, J=9.9 Hz, 1H), 3.91-3.82 (m, 2H), 3.75 (d, J=10.1 Hz, 1H), 3.73-3.52 (m, 5H), 3.50-3.29 (m, 4H), 3.21 (s, 3H), 1.98 (s, 3H), 1.96 (s, 3H), 1.94 (s, 3H), 1.55 (bs, 4H), 1.39-1.30 (m, 2H), 1.26 (d, J=6.6 Hz, 3H); .sup.13C NMR (100 MHz, Acetone-d.sub.6) δ 172.4, 170.5, 170.4, 139.9, 139.8, 139.5, 139.3, 139.1, 137.8, 137.6, 134.3 (2C), 134.0, 133.8, 130.3, 130.0, 129.6, 129.5, 129.3, 129.2 (3C), 129.0, 128.9, 128.8 (2C), 128.7 (2C), 128.6 (3C), 128.5, 128.4 (2C), 128.1, 128.0, 127.1 (3C), 127.0, 126.9, 126.8, 126.6, 126.4, 102.2, 102.0, 101.3, 100.0, 99.4 (2C), 79.0, 78.6, 78.1, 77.0, 76.8, 76.7, 74.6, 73.7, 73.1, 73.0, 71.4, 70.2, 69.9, 69.6, 69.2, 68.7, 68.6, 68.0, 67.4, 67.3, 63.3, 51.8, 49.5, 48.7, 24.2, 23.6 (2C), 23.2, 17.2; HRMS (ESI): Calcd for C.sub.100H.sub.110N.sub.4O.sub.22 [M+Na].sup.+1741.7509, found: 1741.7503.

Example 16A

Synthesis of N-((2R,3S,4R,5S,6R)-2-(((2S,3S,4S,5R,6S)-3-acetamido-2-(((2R,3R,4R,5R,6R)-3-acetamido-2-(((2R,3R,4R,5R,6S)-6-((5-aminopentyl)oxy)-4,5-dihydroxy-2-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)oxy)-5-hydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-4-yl)oxy)-5-hydroxy-6-methyltetrahydro-2H-pyran-4-yl)oxy)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)acetamide (16*)

[0457] ##STR00100##

[0458] To compound 15* (0.019 g, 0.011 mmol) was added MeOH, water, ethyl acetate and acetic acid (3:1:1:0.1-0.3 mL:0.1 mL:0.1 mL:10 μL). Purged the reaction mixture before and after addition of Pd(OH).sub.2 (20%) (0.036 g, 0.052 mmol) with argon for 2-3 min. The reaction mixture was purged with hydrogen and stirred for 24 h under an atmosphere of hydrogen. Filtered off palladium over celite, washed with methanol and 1:1 mixture of MeOH and water, concentrated the filtrate to obtain oil. (LCMS at this point showed no starting material, but only partially debenzylated compounds). Resubjected the crude for a second cycle with the same solvent combination, Pd/C (0.036 g, 10% Pd; 50% water) stirred for 20 h. Filtration as above, followed by LCMS showed only compound. Washed the crude with hexanes and decanted followed by acetone and decantation to obtain the compound 16* as foam (0.0075 g, 79%). [α].sub.D.sup.20=+33.3° (c=0.56, H.sub.2O); IR v.sub.max (film) 3354, 2929, 2860, 1667, 1372, 1096, 1045, 751 cm.sup.−1; .sup.1H NMR (400 MHz, d.sub.2o) δ 5.10 (d, J=2.3 Hz, 1H), 5.05 (d, J=2.5 Hz, 1H), 4.94 (s, 1H), 4.90 (d, J=3.8 Hz, 1H), 4.53-4.48 (m, 2H), 4.45 (dd, J=11.2, 3.7 Hz, 1H), 4.24 (s, 2H), 4.18-4.07 (m, 4H), 4.07-3.92 (m, 5H), 3.93-3.82 (m, 2H), 3.82-3.68 (m, 5H), 3.67-3.52 (m, 2H), 3.46-3.39 (m, 1H), 3.06 (t, J=8.0 Hz, 2H) 2.10 (s, 3H), 2.09 (s, 3H), 2.08 (s, 3H), 1.79-1.66 (m, 4H), 1.56-1.46 (m, 2H), 1.30 (d, J=6.5 Hz, 3H).; .sup.13C NMR (100 MHz, d.sub.2o) δ 175.4, 173.6 (2C), 98.7, 98.3, 98.2, 95.2, 77.2, 76.3, 73.2, 72.6, 71.7, 71.5, 70.3, 69.0, 68.1 (2C), 67.9, 67.6, 66.8 (2C), 60.4, 60.3, 60.2, 53.3, 49.0, 47.6, 39.3, 27.9, 26.4, 22.3, 22.1, 21.9, 21.8, 15.4; HRMS (ESI): Calcd for C.sub.35H.sub.62N.sub.4O.sub.20 [M+Na].sup.+881.3855, found: 881.3790.

[0459] General Procedures for the Preparation of Deletion Sequences:

[0460] Procedure A. Deacylation: To a solution of compound in MeOH (0.06 mL) was added a solution of 0.5 M NaOMe in MeOH (0.013 mL, 6.37 μmol) and the reaction mixture stirred for 2 h at room temperature. Diluted the reaction mixture with MeOH, neutralized with Amberlite 120 H.sup.+resin, filtered and concentrated

[0461] Procedure B. Conversion azide to N-acetamide: To a solution of compound in pyridine (0.6 mL) was added thioacetic acid (0.091 mL, 1.27 mmol) and the reaction mixture stirred at room temperature for 18 h. Removed the solvents under vacuo and azeotroped the reaction mixture twice with toluene to obtain the crude as yellow oil. Purified the crude by flash chromatography using DCM and EtOAc as eluent (0 to 20%).

[0462] Procedure C. Global Deprotection: Dissolved the compound in a mixture of MeOH, water, ethyl acetate and acetic acid (3:1:1:0.1-0.3 mL: 0.1 mL:0.1 mL:10 μL). Purged the reaction mixture before and after addition of Pd(OH).sub.2 (20%) with argon for 2-3 min. The reaction mixture was then purged with hydrogen and stirred for 24 h under an atmosphere of hydrogen. Filtered off palladium over celite, washed with methanol and 1:1 mixture of MeOH and water, and concentrated the filtrate Resubjected the crude for a second cycle with the same solvent combination, Pd/C (10% Pd; 50% water) stirred for 20 h. Filtration as above and washed the crude with hexanes and decanted. Washed the crude with acetone and decantation to obtain the compound as foam after drying.

Example 17A

Synthesis of N-((2S,3R,4R,5R,6R)-2-(((2R,3R,4R,5R,6S)-6-((5-aminopentyl)oxy)-4,5-dihydroxy-2-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)oxy)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)acetamide (17*)

[0463] ##STR00101##

[0464] Following general procedures B, A and C the n-disaccharide deletion sequence 17* was obtained. .sup.1H NMR (400 MHz, d.sub.2o) δ 4.94 (d, J=3.8 Hz, 1H), 4.64 (d, J=8.3 Hz, 1H), 4.15 (s, 1H), 3.98-3.89 (m, 4H), 3.87-3.65 (m, 8H), 3.62-3.47 (m, 1H), 3.03 (t, J=7.4 Hz, 2H), 2.08 (s, 3H), 1.78-1.63 (m, 4H), 1.53-1.42 (m, 2H).; .sup.13C NMR (100 MHz, d.sub.2o) δ 174.9, 102.6, 98.2, 76.9, 74.7, 70.8, 70.1, 69.3, 68.4, 67.8, 67.7, 61.0, 60.6, 52.5, 39.2, 27.9, 26.3 (2C), 22.2; HRMS (ESI): Calcd for C.sub.19H.sub.36N.sub.2O.sub.11 [M+H].sup.+469.2397, found: 469.2383.

Example 18A

Synthesis of N-((2R,3R,4R,5R,6R)-2-(((2R,3R,4R,5R,6S)-6-((5-aminopentyl)oxy)-4,5-dihydroxy-2-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)oxy)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)acetamide (18*)

[0465] ##STR00102##

[0466] Following general procedures B, and C the n-disaccharide deletion sequence 18* was obtained .sup.1H NMR (400 MHz, d.sub.2o) δ 4.83 (d, J=2.9 Hz, 1H), 4.76 (d, J=3.3 Hz, 1H), 4.22 (t, J=6.3 Hz, 1H), 4.03 (dd, J=11.2, 3.7 Hz, 1H), 3.94-3.66 (m, 5H), 3.66-3.48 (m, 5H), 3.51-3.32 (m, 2H), 2.85 (t, J=7.5 Hz, 2H), 1.92 (s, 3H), 1.63-1.46 (m, 4H), 1.38-1.20 (m, 2H).; .sup.13C NMR (100 MHz, d.sub.2o) δ 174.3, 98.2, 98.1, 77.8, 71.6, 70.7, 69.0, 68.2, 68.1, 67.8, 67.0, 60.5, 60.4, 50.1, 39.3, 27.9, 26.4, 22.3, 21.8; HRMS (ESI): Calcd for C.sub.19H.sub.36N.sub.2O.sub.11 [M+H].sup.+469.2397, found: 469.2383.

Example 19A

Synthesis of N-((2R,3S,4S,5S,6S)-2-(((2R,3R,4R,5R,6R)-3-acetamido-2-(((2R,3R,4R,5R,6S)-6-((5-aminopentyl)oxy)-4,5-dihydroxy-2-(hydroxymethyl)tetrahydro-2 H-pyran-3-yl)oxy)-5-hydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-4-yl)oxy)-4,5-dihydroxy-6-methyltetrahydro-2H-pyran-3-yl)acetamide (19*)

[0467] ##STR00103##

[0468] Following general procedures B, and C the β-trisaccharide deletion sequence 19* was obtained .sup.1H NMR (400 MHz, d.sub.2o) δ 4.90 (d, J=3.8 Hz, 1H), 4.88 (d, J=4.0 Hz, 1H), 4.48 (d, J=7.4 Hz, 1H), 4.23 (t, J=6.4 Hz, 1H), 4.13 (dd, J=11.2, 3.7 Hz, 1H), 4.06-3.87 (m, 4H), 3.80 (ddd, J=14.2, 10.6, 3.1 Hz, 2H), 3.73-3.55 (m, 9H), 3.53-3.38 (m, 1H), 2.93-2.89 (m, 2H), 1.98 (s, 3H), 1.95 (s, 3H), 1.68-1.49 (m, 4H), 1.48-1.28 (m, 2H), 1.21 (d, J=6.4 Hz, 3H).; .sup.13C NMR (100 MHz, d.sub.2o) δ 174.9, 174.3, 100.6, 98.4, 98.2, 78.2, 75.7, 71.6, 71.0, 70.8, 70.6, 70.4, 69.0, 68.3, 67.9, 66.3, 60.5, 60.2, 52.5, 48.9, 39.3, 28.0, 26.4, 22.3, 22.2, 22.1, 15.6; HRMS (ESI): Calcd for C.sub.27H.sub.49N.sub.3O.sub.15 [M+Na].sup.+678.3061, found: 678.3003.

Example 20A

Synthesis of N-((2S,3S,4S,5S,6S)-2-(((2R,3R,4R,5R,6R)-3-acetamido-2-(((2R,3R,4R,5R,6S)-6-((5-aminopentyl)oxy)-4,5-dihydroxy-2-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)oxy)-5-hydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-4-yl)oxy)-4,5-dihydroxy-6-methyltetrahydro-2H-pyran-3-yl)acetamide (20*)

[0469] ##STR00104##

[0470] Following general procedures B, and C the a-disaccharide deletion sequence 20* was obtained .sup.1H NMR (400 MHz, d.sub.2o) δ 4.94 (d, J=3.8 Hz, 1H), 4.90 (d, J=2.2 Hz, 1H), 4.75 (d, J=3.8 Hz, 1H), 4.36 (t, J=5.9 Hz, 1H), 4.30 (dd, J=11.1, 3.7 Hz, 1H), 4.06-4.00 (m, 2H), 3.96 (d, J=8.2 Hz, 2H), 3.93-3.78 (m, 5H), 3.74 (d, J=2.5 Hz, 1H), 3.71-3.52 (m, 5H), 3.51-3.38 (m, 1H), 2.91 (t, J=7.5 Hz, 2H), 1.94 (s, 6H), 1.69-1.49 (m, 4H), 1.46-1.25 (m, 2H), 1.14 (d, J=6.5 Hz, 3H).; .sup.13C NMR (100 MHz, d.sub.2o) δ 174.1, 173.6, 98.5, 98.2 (2C), 77.2, 72.9, 71.5, 70.9, 70.4, 69.0, 68.2, 68.1, 67.9, 67.5, 67.1, 60.4, 60.2, 49.4, 49.1, 39.3, 27.9, 26.4, 22.3, 22.1, 21.9, 15.3; HRMS (ESI): Calcd for C.sub.27H.sub.49N.sub.3O.sub.15 [M+Na].sup.+678.3061, found: 678.3068.

Example 21A

Synthesis of N-((2S,3S,4S,5S,6S)-2-((5-aminopentyl)oxy)-4,5-dihydroxy-6-methyltetrahydro-2H-pyran-3-yl)acetamide (21*)

[0471] ##STR00105##

[0472] Following general procedures B, A, and C the FucNAc deletion sequence 21* was obtained .sup.1H NMR (400 MHz, CD.sub.3OD) δ 4.20 (d, J=8.4 Hz, 1H), 3.85-3.67 (m, 2H), 3.56-3.42 (m, 3H), 3.43-3.31 (m, 1H), 3.21 (dt, J=3.1, 1.4 Hz, 1H), 2.80 (t, J=7.5 Hz, 2H), 1.88 (s, 3H), 1.65-1.44 (m, 4H), 1.44-1.26 (m, 2H), 1.17 (d, J=6.4 Hz, 3H); .sup.13C NMR (100 MHz, CD.sub.3OD) δ 174.2, 103.2, 73.4 (2C), 72.0, 70.2, 54.0, 50.0, 40.7, 29.9, 28.3, 23.2, 17.0; HRMS (ESI): Calcd for C.sub.13H.sub.26N.sub.2O.sub.5 [M+Na].sup.+313.1739, found: 313.1728.

Example 22A:

Synthesis of (2S,3R,4S,5S,6S)-5-acetamido-6-(((2S,4aR,6R,7R,8R,8aR)-7-acetamido-6-(((2R,3R,4R,5R,6S)-6-((5-(benzyl((benzyloxy)carbonyl)amino)pentyl)oxy)-2-((benzyloxy)methyl)-4,5-dihydroxytetrahydro-2H-pyran-3-yl)oxy)-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-8-yl)oxy)-4-(((2R,4aR,6R,7S,8R,8aS)-7-acetamido-8-(benzyloxy)-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-6-yl)oxy)-2-methyltetrahydro-2H-pyran-3-yl acetate (22*)

[0473] ##STR00106##

[0474] To a solution of compound 15* (0.035 g, 0.02 mmol) in a mixture of DCM (1.08 mL) and water (0.06 mL) was added DDQ (0.014 g, 0.06 mmol). The reaction mixture was stirred at room temperature for 3 h. The reaction mixture was diluted with water and DCM. The organic layer was washed with sat aq. Na.sub.2S.sub.2O.sub.3, and sat. aq. NaHCO.sub.3 solution, dried over Na.sub.2SO.sub.4, filtered and concentrated to obtain the crude as oil that was further purified by flash chromatography [(silica gel 60, hexanes and ethyl acetate as eluent (0 to 50%)] to obtain the compound 22* as foam (0.021 g, 71%). HRMS (ESI): Calcd for C.sub.80H.sub.96N.sub.4O.sub.23 [M+Na].sup.+1503.6363, found: 1503.6434.

Example 23A

Synthesis of (2R,3aR,4S,6R,7S,7aS)-methyl 7-(((2S,4aR,6R,7R,8R,8aR)-7-acetamido-8-(((2S,3S,4S,5R,6S)-3-acetamido-4-(((2R,4aR,6R,7S,8R,8aS)-7-acetamido-8-(benzyloxy)-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-6-yl)oxy)-5-acetoxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-6-yl)oxy)-4-((5-(benzyl((benzyloxy)carbonyl)amino)pentyl)oxy)-6-((benzyloxy)methyl)-2-methyltetrahydro-3aH-[1,3]dioxolo[4,5-c]pyran-2-carboxylate (23*)

[0475] ##STR00107##

[0476] To a solution of compound 22* (0.010 g, 6.75 μmol) in DCM (0.5 mL) was added methyl 2,2-bis(ethylthio)propanoate (0.0084 g, 0.04 mmol), and 2,4,6-tri-tert-butylpyridine (0.023 g, 0.094 mmol), followed by 4A MS and the reaction mixture stirred at room temperature for 10 min, then cooled to 0° C. and treated with a solution of DMTST (0.0092 g, 0.04 mmol) in DCM (0.2 mL) over a period of 2 h at 0° C. Quenched the reaction mixture with 0.1 mL of Et.sub.3N, filtered and removed the solvents under vacuo. Purified the crude by flash chromatography (DCM, Acetone and MeOH as eluent, 50%) to obtain 5 mg of compound 23* as mixture (R and S) (0.005 g, 47%). HRMS (ESI): Calcd for C.sub.84H.sub.101N.sub.4O.sub.25 [M+H].sup.+1565.6755, found: 1565.6733.

Example 24A

Synthesis of (2S,3aR,4S,6R,7S,7aS)-7-(((2R,3R,4R,5R,6R)-3-acetamido-4-(((2S,3S,4S,5R,6S)-3-acetamido-4-(((2R,3S,4R,5S,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-5-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-5-hydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-4-((5-aminopentyl)oxy)-6-(hydroxymethyl)-2-methyltetrahydro-3aH-[1,3]dioxolo[4,5-c]pyran-2-carboxylic acid (24*)

[0477] ##STR00108##

[0478] To compound 23* (0.005 g, 3.19 μmol) in MeOH (0.5 mL) was added aq. NaOH (0.085 μL 3.75 M) and the reaction mixture was stirred overnight. The solvent was removed under vacuo, diluted with water and cooled to 0° C. The reaction mixture was neutralized with three drops of acetic acid at 0° C. The aqueous was extracted with EtOAc, dried the organic over Na.sub.2SO.sub.4, filtered and concentrated to obtain the crude compound, which by .sup.1H NMR showed no more the acetate and methyl ester peaks. The crude was taken to the next step without furthur purification.

[0479] A solution of the crude compound in a mixture of MeOH, EtOAc and water (3:2:1; 0.3 mL: 0.2 mL: 0.1 mL) was purged with argon for 3 min followed by the addition of Pd(OH).sub.2/C (10%). The reaction mixture was further purged with H.sub.2 and stirred for 48 h under H.sub.2 atmosphere. The reaction mixture was filtered through a PTFE filter and the filtrate was washed with a mixture of 1:1 MeOH: water. The solvents were removed under vacuo to obtain the crude compound that was purified by HPLC to obtain target compound 24* (1 mg, 33%) and its R diastereoisomer as byproduct.

[0480] HRMS (ESI): Calcd for C.sub.38H.sub.64N.sub.4O.sub.22 [M+Na].sup.+951.3910, found: 951.3928.

[0481] Compounds 24*a-24*f constitute further examples according to the present invention that can be obtained following the procedure described for compound 24*:

##STR00109##

[0482] Chemical formula: C.sub.37H.sub.62N.sub.4O.sub.23;

[0483] Molecular weight: 930.3805;

2-(2-Aminoethoxy)ethyl 2-N-acetyl-2-deoxy-β-D-mannopyranosyl-(1→3)-2-N-acetyl-2-deoxy-α-L-fucopyranosyl-(1→3)-2-N-acetyl-2-deoxy-α-D-galactopyranosyl-(1→4)-2,3-O-[1-(S)-(carboxy)-ethylidene]-α-D-galactopyranoside

[0484] ##STR00110##

[0485] Chemical formula: C.sub.36H.sub.58F.sub.2N.sub.4O.sub.22;

[0486] Molecular weight: 936.3511;

3-Amino-2,2-difluoropropyl 2-N-acetyl-2-deoxy-β-D-mannopyranosyl-(1→3)-2-N-acetyl-2-deoxy-α-L-fucopyranosyl-(1→3)-2-N-acetyl-2-deoxy-α-D-galactopyranosyl-(1→4)-2,3-O-[1-(S)-(carboxy)-ethylidene]-α-D-galactopyranoside

[0487] ##STR00111##

[0488] Chemical formula: C.sub.35H.sub.58N.sub.4O.sub.22;

[0489] Molecular weight: 886.3543;

2-Aminoethyl 2-N-acetyl-2-deoxy-β-D-mannopyranosyl-(1→3)-2-N-acetyl-2-deoxy-α-L-fucopyranosyl-(1→3)-2-N-acetyl-2-deoxy-α-D-galactopyranosyl-(1→4)-2,3-O-[1-(S)-(carboxy)-ethylidene]-α-D-galactopyranoside

[0490] ##STR00112##

[0491] Chemical formula: C.sub.40H.sub.57N.sub.5O.sub.23;

[0492] Molecular weight: 985.9880;

2-(5-Aminopentanoyl)-aminoethyl 2-N-acetyl-2-deoxy-β-D-mannopyranosyl-(1→3)-2-N-acetyl-2-deoxy-α-L-fucopyranosyl-(1→3)-2-N-acetyl-2-deoxy-α-D-galactopyranosyl-(1→4)-2,3-O-[1-(S)-(carboxy)-ethylidene]-α-D-galactopyranoside

[0493] ##STR00113##

[0494] Chemical formula: C.sub.42H.sub.71N.sub.5O.sub.24;

[0495] Molecular weight: 1030.0410;

2-(2-(5-Aminopentanoyl)aminoethoxy)ethyl 2-N-acetyl-2-deoxy-β-D-mannopyranosyl-(1→3)-2-N-acetyl-2-deoxy-α-L-fucopyranosyl-(1→3)-2-N-acetyl-2-deoxy-α-D-galactopyranosyl-(14)-2,3-O-[1-(S)-(carboxy)-ethylidene]-α-D-galactopyranoside

[0496] ##STR00114##

[0497] Chemical formula: C.sub.39H.sub.66N.sub.6O.sub.23;

[0498] Molecular weight: 986.9760;

2-(3-Amino-1-aminocarbonyl)aminoethyl 2-N-acetyl-2-deoxy-β-D-mannopyranosyl-(1→3)-2-N-acetyl-2-deoxy-α-L-fucopyranosyl-(1→3)-2-N-acetyl-2-deoxy-α-D-galactopyranosyl-(1→4)-2,3-O-[1-(S)-(carboxy)-ethylidene]-α-D-galactopyranoside

[0499] B. Biological Evaluation

Example 1.B

[0500] Glycan arrays for determination of antibody binding from rabbit SP-4 typing serum and human reference serum 007sp

[0501] Microarray Printing:

[0502] Pyruvated SP4 tetrasaccharide 24* was dissolved at 10 mM in water and diluted to 100 μM and 50 μM in coupling buffer (50 mM sodium phosphate, pH 8.5). Each concentration was spotted in duplicates to CodeLink activated glass slides (Surmodics) alongside a number of related structures and native polysaccharides (see FIG. 4 for printing pattern) using a Scienion S3 microarray printer at 65% relative humidity. The slides were incubated in a humidity saturated chamber overnight. They were washed twice with water and quenched by incubation with 100 mM ethanolamine, 50 mM sodium phosphate pH 9 for one hour at room temperature. They were washed again twice, dried by centrifugation and stored at 4° C. until use.

[0503] Microarray Incubation:

[0504] Slides were blocked by incubation with 1% BSA-PBS at room temperature, washed twice with PBS and dried by centrifugation. A 64 well incubation gasket was attached to the glass slides.

[0505] Sera (rabbit SP4 typing serum (SSI Diagnostica, Denmark) or human reference serum 007sp (Clin. Vaccine Immunol. 2011 18 (10), 1728)) were diluted in 1% BSA-PBS containing no competitors or native SP4 CPS (capsular polysaccharide) or CWPS (S. pneumoniae cell wall polysaccharide) as competitors. The dilutions were incubated for 45min at room temperature and then applied to the microarray according to the attached incubation pattern. After incubation for 1 h at room temperature, wells were washed three times for 5min with PBS containing 0.05% Tween-20 (PBS-T). Incubation with secondary antibodies dilutions in 1% BSA-PBS (for 007sp wells: goat anti-human IgG Fc AlexaFluor 488 (Dianova) 1:400 and goat anti-human IgM AlexaFluor 594 (Invitrogen) 1:400; for rabbit wells: goat anti-rabbit IgG FITC (Abcam) 1:200) was performed for 30min at room temperature in the dark. Wells were washed twice with PBS-T, the gasket was removed and the slides was first rinsed with PBS and then with water. It was dried by centrifugation and the fluorescence was read out using s GenePix 4300a fluorescence reader.

[0506] Results:

[0507] Of all synthetic structures printed to the array, a specific IgG signal in the rabbit typing serum was only observed for the pyruvated SP4 tetrasaccharide 24* (see FIG. 5). This signal could no longer be seen when using native SP4 CPS as competitor, but remained when performing CWPS competition. Compared to the unpyruvated tetrasaccharide 16*, the signal for the pyruvated tetrasaccharide 24* can be inhibited far more efficiently by the native SP4 CPS suggesting a high number of cross-reactive antibodies. CWPS competition has no effect on signal strength.