Vaccines against Streptococcus pneumoniae serotype 5

Abstract

The present invention relates to well-defined synthetic saccharides of general formula (I) that are related to the repeating unit of Streptococcus pneumoniae serotype 5 capsular polysaccharide and conjugates thereof. The conjugates and pharmaceutical compositions containing said conjugates are useful for prevention and/or treatment of diseases associated with Streptococcus pneumoniae, and more specifically against diseases associated with Streptococcus pneumoniae serotype 5. Furthermore, the synthetic saccharides of general formula (I) are useful as marker in immunological assays for detection of antibodies against Streptococcus pneumoniae bacteria. ##STR00001##

Claims

1. A saccharide of general formula (I) ##STR00144## wherein R.sup.1 is selected from R.sup.2, R.sup.4, ##STR00145## R.sup.2 represents ##STR00146## R.sup.3 is selected from H, CH.sub.3, C.sub.2H.sub.5, C.sub.3H.sub.7, C.sub.4H.sub.9 and CF.sub.3; R.sup.4 represents R.sup.6 or ##STR00147## R.sup.5 represents H or ##STR00148## R.sup.6 represents O-L-NH.sub.2; R.sup.7 and R.sup.8 are independently of each other selected from H and OH and cannot be simultaneously H; R.sup.7 and R.sup.8 can form together with the carbon atom to which they are attached to a carbonyl group CO; R.sup.23 is selected from H, C(O)CH.sub.3, C(O)CF.sub.3 and C(O)CCl.sub.3; -L- is selected from: CH.sub.2, (CH.sub.2).sub.2, (CH.sub.2).sub.3, (CH.sub.2).sub.4, (CH.sub.2).sub.5, (CH.sub.2).sub.6, (CH.sub.2).sub.7, (CH.sub.2).sub.8, (CH.sub.2).sub.9, (CH.sub.2).sub.10, CF.sub.2, (CF.sub.2).sub.2, (CF.sub.2).sub.3, (CF.sub.2).sub.4, (CF.sub.2).sub.5, (CF.sub.2).sub.6, (CF.sub.2).sub.7, (CF.sub.2).sub.8, (CF.sub.2).sub.9, (CF.sub.2).sub.10, (CH.sub.2).sub.2O(CH.sub.2).sub.2, CH.sub.2O(CH.sub.2).sub.3, (CH.sub.2).sub.3OCH.sub.2, CH.sub.2O(CH.sub.2).sub.2, (CH.sub.2).sub.2OCH.sub.2, (CH.sub.2).sub.3O(CH.sub.2).sub.2, (CH.sub.2).sub.2O(CH.sub.2).sub.3, (CH.sub.2).sub.4OCH.sub.2, CH.sub.2O(CH.sub.2).sub.4, -L.sup.a-, -L.sup.a-L.sup.e-, -L.sup.a-L.sup.b-L.sup.e-, -L.sup.a-L.sup.b-L.sup.d-L.sup.c-L.sup.e, -L.sup.a-L.sup.d-L.sup.e; wherein -L.sup.a- is selected from: (CH.sub.2).sub.m, (CF.sub.2).sub.m, (CH.sub.2CH.sub.2O).sub.mC.sub.2H.sub.4, (CH.sub.2CH.sub.2O).sub.mCH.sub.2, (CR.sup.10R.sup.11).sub.m, ##STR00149## -L.sup.b- and -L.sup.c- are independently of each other selected from: O, S, NHC(O)NH, NHC(S)NH, NHC(O), C(O)NH, NHC(O)O, NR.sup.9, NR.sup.18, SO.sub.2, ##STR00150## -L.sup.d- represents (CH.sub.2).sub.n, (CF.sub.2).sub.n, (CR.sup.12R.sup.13).sub.n, (CH.sub.2CH.sub.2O).sub.nC.sub.2H.sub.4, (CH.sub.2CH.sub.2O).sub.nCH.sub.2, ##STR00151## -L.sup.e- is selected from: (CH.sub.2).sub.p1, (CF.sub.2).sub.p1, C.sub.2H.sub.4(OCH.sub.2CH.sub.2).sub.p1, CH.sub.2(OCH.sub.2CH.sub.2).sub.p1, (CH.sub.2).sub.p1O(CH.sub.2).sub.p2, (CH.sub.2).sub.p1S(CH.sub.2).sub.p2, (CR.sup.14R.sup.15).sub.p1, (CR.sup.14R.sup.15).sub.p1O(CR.sup.21R.sup.22).sub.p2, (CR.sup.14R.sup.15).sub.p1S(CR.sup.21R.sup.22).sub.p2, ##STR00152## R.sup.9 and R.sup.18 are independently of each other selected from: CH.sub.3, C.sub.2H.sub.5, C.sub.3H.sub.7, and C(O)CH.sub.3; R.sup.10, R.sup.11, R.sup.12, R.sup.13, R.sup.14, R.sup.15, R.sup.16, R.sup.17, R.sup.19, R.sup.20, R.sup.21 and R.sup.22 are independently of each other selected from: H, F, Cl, CH.sub.3, C.sub.2H.sub.5, C.sub.3H.sub.7, C.sub.5H.sub.9, C.sub.6H.sub.13, OCH.sub.3, OC.sub.2H.sub.5, CH.sub.2F, CHF.sub.2, CF.sub.3, C(O)NH.sub.2, SCH.sub.3, SC.sub.2H.sub.5, NHC(O)CH.sub.3, N(CH.sub.3).sub.2 and N(C.sub.2H.sub.5).sub.2; m, n, p1 and p2 are independently of each other an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; and pharmaceutically acceptable salts thereof.

2. The saccharide according to claim 1, wherein R.sup.1 represents ##STR00153## and R.sup.2 and R.sup.3 have the meanings as defined in claim 1.

3. The saccharide according to claim 1, wherein R.sup.4 represents R.sup.6.

4. The saccharide according to claim 1 of general formula (III) ##STR00154## wherein R.sup.3, R.sup.5, R.sup.6 and R.sup.23 have the meanings as defined in claim 1.

5. The saccharide according to claim 1 of the formula 72 ##STR00155## wherein L, R.sup.7, R.sup.8 and R.sup.23 have the meanings as defined in claim 1.

6. The saccharide according to claim 1, wherein R.sup.3 represents H and R.sup.23 represents C(O)CH.sub.3.

7. The saccharide according to claim 1 selected from the group consisting of: 5-amino-pentanyl 2-N-acetyl--L-pneumosaminopyranosyl-(1.fwdarw.2)--D-glucopyranosyluronate (27*), 5-amino-pentanyl 2-N-acetyl--L-pneumosaminopyranosyl-(1.fwdarw.2)--D-glucopyranosyluronate-(1.fwdarw.3)-2-N-acetyl--L-fucosaminopyranosyl-(1.fwdarw.3)-2-N-acetyl--D-fucosaminopyranoside (33*), 5-amino-pentanyl 2-N-acetyl--L-pneumosaminopyranosyl-(1.fwdarw.2)--D-glucopyranosyluronate-(1.fwdarw.3)-2-N-acetyl--L-fucosaminopyranosyl-(1.fwdarw.3)-[-D-glucopyranosyl-(1.fwdarw.4)]-2-N-acetyl--D-fucosaminopyranoside (73*), 5-amino-pentanyl 2-N-acetyl--L-pneumosaminopyranosyl-(1.fwdarw.2)--DD-glucopyranosyluronate-(1.fwdarw.3)-2-N-acetyl--L-fucosaminopyranosyl-(1.fwdarw.3)--D-glucopyranosyl-(1.fwdarw.4)]-2-N-acetyl-3-D-quinovosaminopyranoside (85*), and 5-amino-pentanyl 2-N-acetyl--L-pneumosaminopyranosyl-(1.fwdarw.2)--D-glucopyranosyluronate-(1.fwdarw.3)-2-N-acetyl--L-fucosaminopyranosyl-(1.fwdarw.3)-[-D-glucopyranosyl-(1.fwdarw.4)]-2-acetamido-2,5-dideoxy--D-xylo-hexos-4-uloside (88*).

8. An intermediate compound of formula (IX), 36, 41, 63, 68*, 70 or 70a: ##STR00156## wherein R.sup.24 is selected from ##STR00157## F, Cl, Br, I, SR.sup.28, SeR.sup.29, OPO.sub.3R.sup.30.sub.2; R.sup.25 is selected from N.sub.3, -NBn.sub.2, -NBnCbz; R.sup.26 and R.sup.27 are independently of each other selected from H, -Bn, ##STR00158## Si(CH.sub.3).sub.3, Si(CH.sub.2CH.sub.3).sub.3, C(O)CH.sub.3, C(O)Ph, ##STR00159## or R.sup.26 and R.sup.27 can form together ##STR00160## R.sup.28 is selected from: CH.sub.3, CH.sub.2CH.sub.3, -Ph, ##STR00161## R.sup.29 represents -Ph; R.sup.30 represents CH.sub.2CH.sub.2CH.sub.2CH.sub.3; ##STR00162## wherein L has the meanings as defined in claim 1; ##STR00163## wherein L and R.sup.23 have the meanings as defined in claim 1; ##STR00164## wherein L has the meanings as defined in claim 1; ##STR00165## wherein L has the meanings as defined in claim 1; or ##STR00166## wherein L has the meanings as defined in claim 1.

9. The intermediate according to claim 8 is selected from the group consisting of the saccharides 36, 41, 63, 70, 70a, wherein L is C.sub.5H.sub.10 and/or R.sup.23 is CH.sub.3CO.

10. A conjugate comprising the saccharide according to claim 1.

11. The conjugate according to claim 10 selected from the group consisting of the conjugates of the formulae (X), (XI) and (XII): ##STR00167## wherein x, y and z are independently integer from 1 to 20.

12. The conjugate according to claim 10 selected from the group consisting of 58*a, 58*b, 58*c and 87*: ##STR00168##

13. A method for raising a protective immune response in a human or animal host comprising administering to the human or animal host a saccharide according to claim 1 or a conjugate comprising the saccharide.

14. A method for prevention and/or treatment of diseases associated with bacteria containing N-acetyl-L-pneumosamine in their capsular polysaccharide comprising administering to a patient a saccharide according to claim 1 or a conjugate comprising the saccharide.

15. The method according to claim 14, wherein the bacteria is Streptococcus pneumoniae serotype 5.

16. The method according to claim 14, wherein the diseases associated with the bacteria are selected from pneumonia, meningitis, otitis media, bacteremia and acute exacerbation of chronic bronchitis, sinusitis, arthritis and conjunctivitis.

17. A pharmaceutical composition comprising the conjugate according to claim 10 together with at least one pharmaceutically acceptable cryoprotectant, lyoprotectant, excipient and/or diluent.

18. The pharmaceutical composition according to claim 17 further comprising at least one of antigens of other Streptococcus pneumoniae serotypes.

19. An immunological assays comprising a saccharide of claim 1 conjugated to a solid support, wherein the assay is configured for detection of antibodies against bacteria containing N-acetyl-L-pneumosamine in their capsular polysaccharide.

20. A pharmaceutical composition comprising the saccharide according claim 1 together with at least one pharmaceutically acceptable cryoprotectant, lyoprotectant, excipient and/or diluent.

21. The saccharide according to claim 1, wherein the linker -L- represents (CH.sub.2).sub.m, wherein m is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1 shows the chemical structure of the S. pneumoniae serotype 5 repeating unit.

(2) FIG. 2 provides examples of commercially available interconnecting molecules according to the present invention.

(3) FIG. 3 provides examples of functional group X of the interconnecting molecule according to the present invention.

(4) FIG. 4 shows the results of the glycan array screening of S. pneumoniae serotype 5 rabbit typing serum. A) Representative well of rabbit typing serum (dilution 1:1000); B) Representative well of rabbit typing serum (dilution 1:1000) pre-incubated with SP5 CPS (5 g/mL); C) Table listing the positions 1-11 of respective glycans. Samples 14* and 20* were impure.

(5) FIG. 5 shows the results of the glycan array screening of human pneumococcal CPS vaccine recipients' pooled serum. A) Representative well of human pneumococcal serum (dilution 1:100); B) Representative well of human pneumococcal serum (dilution 1:100) pre-incubated with SP5 CPS (5 g/mL); C) Table listing the positions 1-11 of respective glycans. Samples 14* and 20* were impure.

(6) FIG. 6: A) Chemical structure of synthetic SP-5 tetrasaccharide 33*; B) Tetrasaccharide 33* was conjugated with CRM.sub.197 by PNP based chemistry and resolved on 10% SDS-PAGE along with recombinant CRM.sub.197 and stained with Coomassie brilliant blue R250. The molecular weight marker is shown in the middle of SDS-PAGE; C) The protein amount was estimated using the standard curve plotted with known concentration of BSA (BCA: Bicinchoninic Acid).

(7) FIGS. 7: A and B: The tetrasaccharide 33* and pentasaccharide 73* of ST5 were conjugated with CRM.sub.197 by PNP based chemistry and resolved on 10% SDS-PAGE along with recombinant CRM.sub.197 and stained with Coomassie brilliant blue R250 respectively. The molecular weight marker is shown in the extreme left of SDS-PAGE; C and D: Matrix-assisted laser desorption/ionization (MALDI) analysis was carried out to measure the average molecular size of ST5 tetrasaccharide 33* and pentasaccharide 73* conjugates at pH 8.0 respectively. The recombinant CRM.sub.197 was used as standard.

(8) FIG. 8: Microarray with ST5 type sera

(9) A) Microarray slide printing pattern. (B) Microarray slide was incubated with rabbit type 5 sera (Statens Serum Institute, Denmark) at dilution 1 in 100. Microarray analysis suggested that type sera recognized synthetic structures along with native CPS-5 polysaccharide. (C) Structure description printed on the slide.

(10) FIG. 9: Microarray with rabbit anti-ST5 synthetic conjugates serum

(11) (A) Immunization pattern and sera collection (printing pattern are the same in all studies). Rabbit were immunized as prime boost manner at day 0, 14 and 28. Preimmune (day 0) and hyperimmune serum (day 14, 21 and 35) was collected from individual rabbits. (B and D) Microarray slides were incubated with pooled rabbit sera (day 35) raised against tetrasaccharide and pentasaccharide conjugates to the slides. Both group of hyperimmune sera exhibited cross-reactivity with native polysaccharide. (C) Printing pattern of microarray slides.

(12) FIG. 10: End point titer analysis.

(13) Immunization with ST5 glycan conjugates induces high antibody titers in rabbits. Three rabbits in each group (female ZIKA rabbits, 10-12 weeks, 2.5-3 kg) were immunized subcutaneously with 3 doses of 10 g of tetrasaccharide and pentasaccharide conjugate (in alum) at day 0, 14 and 28. The immune response were analyzed and plotted in terms of fold change at every time point (A). The endpoint titer (total IgG) was determined in hyperimmune serum (day 35) from individual rabbit (B). Each bar represents the individual rabbit antibody titer. Antibody titers were plotted as the reciprocal of the highest dilution that gave a reading minus the absorbance value obtained with the preimmune sera (day 0; 1 in 100 dilutions). The antibody response between the groups were also analyzed (C).

(14) FIG. 11: Surface staining of anti-ST5 glycan conjugates with pneumococcal strain. Surface binding of anti-ST5 glycan conjugates sera with serotype 5 strain were analyzed by flow cytometry. The solid grey histogram represents the negative control (bacteria+secondary antibody), doted black represent the day 0. Black and gray histograms represent the tetrasaccharide and pentasaccharide respectively.

(15) FIG. 12 Anti-ST5 (tetrasaccharide) antibodies promote the phagocytosis of pneumococci.

(16) Differentiated HL-60 cells were incubated with serotype 5 bacteria pretreated with anti-ST5 (tetrasaccharide) or preimmune sera, and pneumococcal survival was assessed after 45 min. Percent killing of pneumococci was calculated based on viable pneumococcal colonies obtained relative to no sera control. This is the represent values one out of three independent experiments done in triplicates. Values represent meanSE

(17) 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.

(18) 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

(19) Chemical Synthesis

Abbreviations

(20) NIS: N-iodosuccinimide;

(21) TfOH: triflic acid;

(22) h: hour;

(23) DCM: dichloromethane;

(24) TLC: thin layer chromatography;

(25) rt: room temperature;

(26) EtOAc: ethyl acetate;

(27) MS: molecular sieves;

(28) TMS: trimethylsilyl;

(29) Tempo: 2,2,6,6-tetramethyl-1-piperidinyloxy, free radical;

(30) BAIB: [bis(acetoxy)iodo]benzene;

(31) TMSOTf: trimethylsilyl triflate;

(32) TBAF: tetrabutylammonium fluoride;

(33) TDS: dimethylthexylsilyl;

(34) DDQ: 2,3-dichloro-5,6-dicyano-1,4-benzoquinone;

(35) Et.sub.3N: triethylamine;

(36) DBU: 1,8-diazabicycloundec-7-ene;

(37) Nap: 2-naphthylmethyl;

(38) equiv: equivalents;

(39) sat.: saturated;

(40) aq.: aqueous;

(41) Hex: hexanes;

(42) TMSN.sub.3: trimethylsilyl azide;

(43) PMBCl: p-methoxybenzyl chloride;

(44) THF: tetrahydrofuran;

(45) DMAP: 4-dimethylaminopyridine;

(46) TBAI: tetrabutylammonium iodide;

(47) TBAB: tetrabutylammonium bromide;

(48) Bu.sub.2SnO: dibutyltin oxide;

(49) CAN: cerium ammonium nitrate;

(50) BnBr: benzylbromide;

(51) BH.sub.3.THF: borane tetrahydrofuran complex;

(52) DMF: dimethylformamide;

(53) UV: ultraviolet;

(54) NMR: nuclear magnetic resonance;

(55) MALDI-TOF: matrix-assisted laser desorption/ionization-time of flight spectrometry;

(56) HRMS: high resolution mass spectroscopy;

(57) ESI: electrospray ionization;

(58) MeCN: acetonitrile;

(59) IR: infrared;

(60) EDC: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide;

(61) Et.sub.2O: diethyl ether;

(62) NaOMe: sodium methoxide;

(63) OTCA: trichloroacetimidate;

(64) General Information for Chemical Synthesis

(65) Commercial reagents were used without further purification except where noted. Solvents were dried and redistilled prior to use in the usual way. All reactions were performed in oven-dried glassware under an inert atmosphere unless noted otherwise. Analytical thin layer chromatography (TLC) was performed on Kieselgel 60 F254 aluminium plates precoated with a 0.25 mm thickness of silica gel. The TLC plates were visualized with UV light and by staining with Hanessian solution (ceric sulfate and ammonium molybdate in aqueous sulfuric acid) or sulfuric acid-ethanol solution. Column chromatography was performed on Fluka Kieselgel 60 (230-400 mesh). Optical rotations (OR) were measured with a Schmidt & Haensch UniPol L1000 polarimeter at a concentration (c) expressed in g/100 mL. .sup.1H and .sup.13C NMR spectra were measured with a Varian 400-MR or Varian 600 spectrometer with Me.sub.4Si as the internal standard. NMR chemical shifts () were recorded in ppm and coupling constants (J) were reported in Hz. High-resolution mass spectra (HRMS) were recorded with an Agilent 6210 ESI-TOF mass spectrometer at the Freie Universitt Berlin, Mass Spectrometry Core Facility.

(66) General Procedure (A) for Removal of the TDS Group at the Anomeric Position.

(67) Anomeric TDS-protected starting material (1.0 equiv) was dissolved in THF (reaction concentration at 0.15 M) and cooled to 0 C. A solution of TBAF (1 M in THF, 10 equiv) and AcOH (12 equiv) was added, warmed to room temperature and stirred for 3 h. The reaction mixture was diluted with EtOAc, washed with 0.1 N HCl, sat. aq. NaHCO.sub.3 and brine. The organic phases were dried over MgSO.sub.4 and concentrated. Column chromatography on silica gel (hexanes/EtOAc) afforded the corresponding lactol as a mixture of and anomers.

(68) General Procedure (B) for NIS/TfOH-Mediated Glycosylation of Thio- and Selenoglycosides.

(69) The acceptor (1.0 to 2.0 equiv) and thio- or selenoglycoside (1.0 to 1.3 equiv) were co-evaporated with toluene three times and dried in vacuo. The residue was dissolved in DCM or MeCN (reaction concentration at 80 to 120 mM) together with freshly activated molecular sieves (4 ) and NIS (1.2 equiv with regard to the thio- or selenoglycoside). The mixture was cooled and TfOH (0.1 equiv with regard to the thio- or selenoglycoside) was added. The reaction was stirred for 1 h, then quenched by the addition of NEt.sub.3 and diluted with DCM. The organic layer was washed with sat. aq. Na.sub.2S.sub.2O.sub.3 followed by sat. aq. NaHCO.sub.3, dried over MgSO.sub.4 and concentrated. Column chromatography (hexanes/EtOAc) afforded the pure product.

(70) General Procedure (C) for Glycosyl-Trichloroacetimidate Synthesis with K.sub.2CO.sub.3.

(71) To a solution of the lactol (1.0 equiv) in DCM (reaction concentration at 0.5 M) trichloroacetonitrile (10 equiv) and K.sub.2CO.sub.3 (1.7 equiv) were added and stirred for 3 h at room temperature. The crude product was concentrated and purified by column chromatography on silica gel (hexanes/EtOAc) to afford pure product.

(72) General Procedure (D) for Glycosyl-Trichloroacetimidate Synthesis with DBU.

(73) To a solution of the lactol (1.0 equiv) in DCM (reaction concentration at 0.5 M) at 0 C. trichloroacetonitrile (10 equiv) and DBU (1 drop) were added and stirred for 1 h at 0 C. The crude product was concentrated and purified by column chromatography on silica gel (hexanes/EtOAc) to afford pure product.

(74) General Procedure (E) for TMSOTf-Mediated Glycosylation of Glycosyl-Imidates.

(75) The acceptor (1.0 to 2.0 equiv) and glycosyl-trichloroacetimidate (1.0 to 1.3 equiv) were co-evaporated with toluene three times and dried in vacuo. The residue was dissolved in DCM (reaction concentration at 80 to 120 mM) and freshly activated molecular sieves (4 ) were added. The mixture was cooled to 30 C. and TMSOTf (0.1 equiv) was added. The reaction was brought to 20 C., then quenched by the addition of NEt.sub.3 and concentrated under reduced pressure. Column chromatography (hexanes/EtOAc) afforded the pure product.

(76) General Procedure (F) for Thioacetic Acid Based Azide Reduction.

(77) To a solution of azide starting material in pyridine (1 mL) thioacetic acid (0.5 mL) was added and stirred for 12 to 48 h. The reaction mixture was concentrated and purified by column chromatography on silica gel (hexanes/acetone) to afford the acetamide product.

(78) General Procedure (G) for Removal of the Nap Protecting Group.

(79) To a mixture of naphthylated starting material (1 equiv) in a mixture of DCM/phosphate-buffer (10:1 (v:v), 7 mM, pH 7.2, reaction concentration at 4 mM or DCM/H.sub.2O (10:1) at 0 C. DDQ (2-3 equiv) was added portionwise over 1.5 h. The reaction mixture was warmed to room temperature and stirred for further 1.5 to 5 h. The mixture was diluted with sat. aq. NaHCO.sub.3 solution, extracted with DCM and the organic layer dried over MgSO.sub.4 and concentrated. Column chromatography on silica gel (hexanes/EtOAc) afforded the pure product.

(80) General Procedure (H) for TEMPO-Mediated Oxidation.

(81) To a mixture of the primary alcohol (1.0 equiv) in DCM/H.sub.2O ((2.5:1, v:v), reaction concentration at 7 mM) at 0 C. was added TEMPO (0.2 equiv) and BAIB (5.0 equiv). The reaction mixture was warmed to room temperature, stirred for 2 h and then diluted with H.sub.2O and extracted with DCM. The organic phases were dried over Na.sub.2SO.sub.4 and concentrated. Size exclusion chromatography on Sephadex LH-20 (CHCl.sub.3/MeOH=1:1) afforded the product.

(82) General Procedure (I) for Hydrogenolysis.

(83) A solution of starting material (reaction concentration at 4 to 8 mM) in a mixture of EtOH/EtOAc/H.sub.2O/AcOH was purged with Ar. After that 10% Pd/C was added and the solution purged with H.sub.2 for 10 min, then stirred under an H.sub.2 atmosphere for 12 h, filtered and concentrated. The crude product was dissolved in H.sub.2O, subjected to reversed phase solid phase extraction (RP SPE) (Waters Sep-Pak, C18) and lyophilized. When necessary, size exclusion chromatography on Sephadex LH-20 (MeOH) was performed.

Example 1: Synthesis of phenyl 2-azido-2-deoxy-1-seleno--D-fucopyranoside (1*)

(84) ##STR00060##

(85) To a solution of the fucal (Bedini, E. et. al. Synlett 2006, 6, 825-830) (4.6 g, 21.6 mmol) and Ph.sub.2Se.sub.2 (6.75 g, 21.6 mmol) in DCM (70 mL) at 30 C. was added PhI(OAc).sub.2 (6.96 g, 21.6 mmol). Then, TMSN.sub.3 (5.74 mL, 43.2 mmol) was added dropwise to the resulting solution. The reaction was warmed to 10 C. After 4 h at 10 C. the reaction was complete. The reaction was mixture was allowed to warm to room temperature and washed with NaHCO.sub.3 saturated solution followed by brine. The solvent was evaporated and the impure was dissolved in MeOH (190 mL). NaOMe 0.5 M (4.75 mmol, 9.5 mL) solution was added dropwise to the mixture at room temperature. The reaction was stirred at room temperature for 1 h. Amberlite was added and let stir until the pH decreased to 5. The reaction mixture was filtered and the solvent evaporated. The residue was purified by silica-gel chromatography (DCM 100% to DCM:MeOH:Acetone 97:1.5:1.5). The yellow solid obtained was washed with hexanes to give diol 1* as a white solid (4.24 g, 12.9 mmol, 60% yield). [].sub.D.sup.20=241.6 (c=1.00, CHCl.sub.3); IR .sub.max (film) 3321, 2925, 2105, 1578, 1475, 1438, 1093, 1059, 739 cm.sup.1; .sup.1H NMR (400 MHz, CD.sub.3OD) 7.71-7.47 (m, 2H), 7.35-7.09 (m, 3H), 5.90 (d, J=5.4 Hz, 1H), 4.28 (q, J=6.6 Hz, 1H), 4.00 (dd, J=9.9, 5.3 Hz, 1H), 3.70 (ddd, J=6.6, 4.5, 2.2 Hz, 2H), 1.13 (d, J=6.5 Hz, 3H). .sup.13C NMR (101 MHz, CD.sub.3OD) 134.5, 128.6, 127.3, 85.5, 71.5, 71.3, 69.2, 61.5, 15.0. HRMS (ESI+) Calcd for C.sub.12H.sub.15O.sub.3N.sub.3SeNa.sup.+ [M+Na].sup.+ 352.0171, found 352.0179.

Example 2: Synthesis of phenyl 2-azido-2-deoxy-3-O-p-methoxybenzyl-1-seleno--D-fucopyranoside (2*)

(86) ##STR00061##

(87) Diol 2* (2.5 g, 7.62 mmol) was co-evaporated with dry toluene twice and let dry under high vacuum for 30 min. Then, dry toluene (80 mL) was added, followed by Bu.sub.2SnO (2.84 g, 11.43 mmol) and 4 A MS. The reaction was stirred for 1 hour under reflux. The reaction was cooled to 40 C.; PMBCl (3.11 mL, 22.85 mmol) and TBAB (3.68 g, 11.43 mmol) were added and left stir overnight at rt. In the morning the reaction was complete. The reaction was filtered and the solvent evaporated. The residue was purified by silica-gel chromatography (EtOAC in Hex 10 to 20%). Alcohol 2* was obtained as a yellow oil (3.09 g, 6.89 mmol, 90%). [].sub.D.sup.20=+177.4 (c=0.77, CHCl.sub.3); IR .sub.max (film) 3493, 2935, 2111, 1613, 1514, 1249, 1091, 740 cm.sup.1; .sup.1H NMR (400 MHz, CDCl.sub.3) 7.79-7.47 (m, 2H), 7.41-7.12 (m, 5H), 6.92 (d, J=8.7 Hz, 2H), 5.88 (d, J=5.4 Hz, 1H), 4.69 (d, J=11.0 Hz, 1H), 4.63 (d, J=11.0 Hz, 1H), 4.29 (q, J=6.5 Hz, 1H), 4.15 (dd, J=10.2, 5.3 Hz, 1H), 3.89-3.84 (m, 1H), 3.82 (s, 3H), 3.69 (dd, J=10.2, 3.1 Hz, 1H), 2.36 (s, 1H), 1.26 (d, J=6.6 Hz, 3H). .sup.13C NMR (101 MHz, CDCl.sub.3) 159.7, 134.4, 129.8, 129.1, 129.0, 128.5, 127.8, 114.1, 85.2, 78.9, 71.8, 68.6, 68.5, 60.1, 55.3, 16.1. HRMS (ESI+) Calcd for C.sub.20H.sub.23O.sub.4N.sub.3SeNa.sup.+ [M+Na].sup.+ 472.0746, found 472.0755.

Example 3: Synthesis of phenyl 2-azido-2-deoxy-3-O-p-methoxybenzyl-4-O-(2-naphthalenylmethyl)-1-seleno--D-fucopyranoside-2-(bromomethyl)-naphthalene (3*)

(88) ##STR00062##

(89) 2-(bromomethyl)-naphthalene (2.8 g, 12.67 mmol) was added to a solution of alcohol 2* (2.84 g, 6.33 mmol) in DMF:THF (1:1, 64 mL). Then, the mixture was cooled to 0 C. and NaH 60% (304 mg, 7.60 mmol) was added. The reaction was warmed to room temperature and let stir for 1 h. The reaction was not completed. Then, it was cooled to 0 C. again and 100 mg of NaH were added. In 1 h the reaction was completed. The reaction mixture was cooled to 0 C., MeOH was added and the reaction warmed to room temperature. The reaction mixture was diluted with ether, washed with HCl, NaHCO.sub.3 and brine. The solvent was evaporated and the residue purified by silica-gel chromatography (EtOAC in Hex 5 to 10%). Product 3* was obtained as a colorless oil (2.56 g, 4.35 mmol, 69%). [].sub.D.sup.20=151.28 (c=2.00, CHCl.sub.3); IR .sub.max (film) 2896, 2109, 1612, 1513, 1249, 1101, 1065, 820, 740 cm.sup.1; .sup.1H NMR (400 MHz, CDCl.sub.3) 7.94-7.75 (m, 3H), 7.71 (s, 1H), 7.63-7.53 (m, 2H), 7.52-7.42 (m, 3H), 7.40-7.31 (m, 2H), 7.29-7.20 (m, 3H), 7.00-6.84 (m, 2H), 5.94 (d, J=5.2 Hz, 1H), 5.09 (d, J=11.7 Hz, 1H), 4.78 (d, J=11.7 Hz, 1H), 4.74 (d, J=11.1 Hz, 1H), 4.70 (d, J=11.1 Hz, 1H), 4.48-4.30 (m, 1H), 4.22 (q, J=6.5 Hz, 1H), 3.84 (s, 1H), 3.77-3.66 (m, 1H), 1.13 (d, J=6.5 Hz, 3H). .sup.13C NMR (101 MHz, CDCl.sub.3) 159.5, 135.6, 134.3, 133.1, 133.0, 129.6, 129.0, 128.7, 128.1, 127.9, 127.7, 127.6, 126.9, 126.3, 126.1, 125.9, 114.0, 85.6, 80.4, 75.7, 74.9, 72.4, 69.4, 60.9, 55.3, 16.6. HRMS (ESI+) Calcd for C.sub.31H.sub.31O.sub.4N.sub.3SeNa.sup.+ [M+Na].sup.+ 612.1373, found 612.1371.

Example 4: Synthesis of 2-azido-2-deoxy-3-O-p-methoxybenzyl-4-O-(2-naphthalenylmethyl)-,-D-fucopyranosyl trichloroacetimidate (4*)

(90) ##STR00063##

(91) Selenide compound 3* (730 mg, 1.24 mmol) was dissolved in THF:H.sub.2O (1.7:1, 38 mL) and NIS (558 mg, 2.48 mmol) added. The reaction was stirred at room temperature for 2 h and quenched with NaS.sub.2O.sub.3. Then, the reaction mixture was diluted with CH.sub.2Cl.sub.2 and washed with NaHCO.sub.3 and brine. The solvent was evaporated and the residue purified by silica-gel chromatography (EtOAc in Hex 20%). A colorless oil was obtained in 94% yield (524 mg, 1.17 mmol). The product was dissolved in DCM (12 mL) and cooled to 0 C. Trichloroacetonitrile (1.16 mL, 11.57 mmol) and DBU (0.012 mmol, 2 L) were added. After 1 h stirring at 0 C. the reaction was not complete. Then, trichloroacetonitrile (1 mL) was added. One hour later the reaction was complete. The solvent was evaporated and the residue purified by silica-gel chromatography (EtOAc in Hex 20%). Trichloroacetimidate 4* was obtained as mixture of alpha and beta anomers (:, 1:3) in quantitative yield (700 mg, 1.18 mmol). Characterization is given for the beta anomer. [].sub.D.sup.20=8.69 (c=1.99, CHCl.sub.3); IR .sub.max (film) 2934, 2113, 1675, 1513, 1059, 821, 796 cm.sup.1; .sup.1H NMR (400 MHz, CDCl.sub.3) 8.63 (s, 1H), 7.98-7.78 (m, 3H), 7.73 (s, 1H), 7.59-7.43 (m, 3H), 7.34 (d, J=8.7 Hz, 2H), 6.92 (d, J=8.7 Hz, 2H), 5.53 (d, J=8.5 Hz, 1H), 5.10 (d, J=11.8 Hz, 1H), 4.86 (d, J=11.8 Hz, 1H), 4.70 (s, 2H), 4.07 (dd, J=10.2, 8.6 Hz, 1H), 3.83 (s, 3H), 3.68-3.52 (m, 2H), 3.45 (dd, J=10.3, 2.7 Hz, 1H), 1.23 (d, J=6.4 Hz, 3H). .sup.13C NMR (101 MHz, CDCl.sub.3) 161.5, 159.5, 135.4, 133.1, 133.0, 129.6, 129.5, 128.1, 127.9, 127.7, 127.4, 126.6, 126.1, 126.0, 114.0, 97.2, 92.5, 80.7, 74.8, 74.5, 72.6, 71.9, 62.5, 55.3, 29.7, 16.9. HRMS (ESI+) Calcd for C.sub.27H.sub.27O.sub.4N.sub.4Cl.sub.3Na.sup.+ [M+Na].sup.+ 617.0912, found 617.0930.

Example 5: Synthesis of N-(benzyl)benzyloxycarbonyl-5-amino-pentanyl 2-azido-2-deoxy-3-O-p-methoxybenzyl-4-O-(2-naphthalenylmethyl)--D-fucopyranoside (5*)

(92) ##STR00064##

(93) Imidate 4* (538 mg, 0.91 mmol) and the linker N-(benzyl)benzyloxycarbonyl-5-amino-1-pentanol (593 mg, 1.8 mmol) were co-evaporated with dry toluene and dried in high vacuum. The residue was dissolved in acetonitrile and cooled to 15 C. TMSOTf (16 L, 0.09 mmol) was added and the reaction was stirred for 1 h. The reaction was quenched with triethylamine and the solvent evaporated. The residue was purified by silica-gel chromatography (EtOAc in Hex 15 to 20%) to give monosaccharide 5* as a colorless oil in 74% yield (510 mg, 0.672 mmol). [].sub.D.sup.20=21.34 (c=2.10, CHCl.sub.3); IR .sub.max (film) 2935, 2110, 1697, 1513, 1248, 1068, 819, 699 cm.sup.1; .sup.1H NMR (400 MHz, CDCl.sub.3) 7.90-7.77 (m, 3H), 7.72 (s, 1H), 7.58-7.43 (m, 3H), 7.38-7.14 (m, 12H), 6.91 (d, J=8.6 Hz, 2H), 5.18 (d, J=12.1 Hz, 1H), 5.07 (d, J=11.9 Hz, 1H), 4.84 (d, J=11.9 Hz, 1H), 4.73-4.60 (m, 1H), 4.50 (d, J=7.6 Hz, 1H), 4.14 (t, J=8.7 Hz, 1H), 3.93-3.71 (m, 2H), 3.53 (d, J=2.4 Hz, 1H), 3.44-3.35 (m, 1H), 3.32-3.15 (m, 1H), 1.66-1.47 (m, 1H), 1.41-1.26 (m, 1H), 1.19 (d, J=6.4 Hz, 1H). .sup.13C NMR (101 MHz, CDCl.sub.3) 137.9, 135.7, 133.1, 133.0, 129.8, 129.5, 128.5, 128.5, 128.0, 127.9, 127.8, 127.7, 127.2, 126.7, 126.0, 125.9, 113.9, 102.3, 80.7, 74.7, 74.6, 72.5, 70.5, 69.6, 67.1, 63.1, 55.3, 50.5, 50.2, 47.1, 46.2, 29.2, 27.9, 27.5, 23.2, 17.0. HRMS (ESI+) Calcd for C.sub.45H.sub.50O.sub.7N.sub.4Na.sup.+ [M+Na].sup.+ 781.3577, found 781.3590.

Example 6: Synthesis of N-(benzyl)benzyloxycarbonyl-5-amino-pentanyl 2-azido-2-deoxy-4-O-(2-naphthalenylmethyl)--D-fucopyranoside (6*)

(94) ##STR00065##

(95) PMB protected compound 5* (510 mg) was dissolved in acetone (10 mL) and H.sub.2O (1.1 mL), then 700 mg of CAN was added (solid). After that, a solution of CAN (700 mg) in acetone (1.7 mL) and H.sub.2O (0.2 mL) was added over 70 min. After 10 min the reaction was poured in NaHCO.sub.3 and extracted with CH.sub.2Cl.sub.2. The solvent was evaporated and the residue purified by silica-gel chromatography (EtOAc in Hex 20%). Alcohol 6* was obtained was a yellow oil in 73% yield (315 mg, 0.493 mmol). [].sub.D.sup.20=1.73 (c=2.18, CHCl.sub.3); IR .sub.max (film) 3440, 3031, 2935, 2109, 1694, 1068, 698 cm.sup.1; .sup.1H NMR (400 MHz, CDCl.sub.3) 7.89-7.74 (m, 4H), 7.62-7.43 (m, 3H), 7.41-7.21 (m, 9H), 7.17 (d, J=6.9 Hz, 1H), 5.18 (d, J=13.1 Hz, 2H), 4.97 (d, J=11.8 Hz, 1H), 4.90 (d, J=11.8 Hz, 1H), 4.50 (d, J=8.7 Hz, 2H), 4.18 (t, J=8.6 Hz, 1H), 3.95-3.80 (m, 1H), 3.64-3.37 (m, 5H), 3.23 (m, 2H), 2.31 (s, 1H), 1.69-1.46 (m, 4H), 1.43-1.18 (m, 2H), 1.31 (d, J=6.5 Hz, 3H). .sup.13C NMR (101 MHz, CDCl.sub.3) 137.9, 135.3, 133.2, 133.1, 128.5, 128.4, 128.0, 127.9, 127.8, 127.7, 127.2, 127.1, 126.2, 126.1, 126.1, 102.2, 78.2, 75.9, 73.0, 70.8, 69.7, 67.1, 64.7, 50.5, 50.2, 47.1, 46.2, 29.2, 27.9, 27.4, 23.2, 17.0. HRMS (ESI+) Calcd for C.sub.37H.sub.42O.sub.6N.sub.4Na.sup.+ [M+Na].sup.+ 661.3002, found 661.3015.

Example 7: Synthesis of phenyl 2-azido-3,4-di-O-benzyl-2-deoxy-1-seleno--L-pneumopyranoside (7*)

(96) ##STR00066##

(97) To a impure mixture containing approx. 25% of 2-azido-2-deoxy-1-seleno--L-pneumopyranoside (192 mg) in DMF (5 mL) at 0 C., BnBr (0.21 mL, 1.8 mmol) and NaH (31 mg, 1.3 mmol) were added. After 3 h the reaction was quenched with MeOH at 0 C. and diluted with Et.sub.2O. The organic layer was washed with 0.1 M aq. HCl solution, sat. aq. NaHCO.sub.3 and brine. The organic layers were dried over MgSO.sub.4 and concentrated. Column chromatography on silica gel (hexanes/ethyl acetate) afforded 7* (66 mg, 0.13 mmol, 22%). [].sub.D.sup.20=72.2 (c=1.6, CHCl.sub.3); IR v.sub.max (film) 3062, 3031, 2933, 2869, 2106, 1578, 1496, 1477, 1454, 1438, 1361, 1279, 1202, 1162, 1105, 1067 cm.sup.1; .sup.1H-NMR (400 MHz, CDCl.sub.3) 7.58-7.23 (m, 15H), 5.80 (d, J=1.5, 1H), 5.02 (J=11.8, 1H), 4.81-4.64 (m, 3H), 4.24-4.10 (m, 2H), 3.99-3.90 (m, 1H), 3.71-3.66 (m, 1H), 1.24 (d, J=6.5, 3H); .sup.13C-NMR (100 MHz, CDCl.sub.3) 138.4, 137.4, 133.9, 129.4, 129.1, 128.8, 128.6, 128.3, 128.2, 128.0, 127.8, 127.7, 83.9, 77.5, 75.4, 75.0, 71.4, 70.6, 60.0, 16.7; HRMS (ESI): Calcd for C.sub.26H.sub.27N.sub.3O.sub.3SeNa.sup.+ [M+Na].sup.+ 532.1115, found 532.1112.

Example 8: Synthesis of 2-azido-3,4-di-O-benzyl-2-deoxy--L-pneumopyranosyl trichloroacetimidate (8*)

(98) ##STR00067##

(99) To a solution of 7* (30 mg, 59 mol) in THF (1.25 mL) and H.sub.2O (0.75 mL) was added NIS (27 mg, 180 mol) and stirred for 2 h. The reaction was diluted with DCM and the organic layer washed with sat. aq. Na.sub.2S.sub.2O.sub.3 followed by sat. aq. NaHCO.sub.3, dried over MgSO.sub.4 and concentrated. Column chromatography (hexanes/EtOAc) afforded the free lactol (20 mg, 54 mol, 92%) as a mixture of and anomers. IR v.sub.max (film) 3413, 3031, 2872, 2110, 1496, 1454, 1360, 1306, 1176, 1109, 1053, 1027 cm.sup.1; .sup.1H-NMR (400 MHz, CDCl.sub.3) 7.59-7.20 (m, 10H), 5.20 (d, J=1.6 Hz, 0.5H), 4.99 (d, J=11.7 Hz, 1H), 4.81-4.64 (m, 3H), 4.56 (d, J=1.8 Hz, 0.5H), 4.02 (t, J=3.4 Hz, 1H), 3.67 (t, J=3.4 Hz, 0.5H), 3.65-3.63 (m, 0.5H), 3.60-3.55 (m, 0.5H), 3.44 (qd, J=6.3, 1.1 Hz, 0.5H), 1.26 (d, J=6.4 Hz, 1.5H), 1.23 (d, J=6.6 Hz, 1.5H); .sup.13C-NMR (100 MHz, CDCl.sub.3) 138.4, 138.1, 137.8, 137.3, 129.0, 128.8 (2C), 128.6, 128.4, 128.3, 128.2, 127.9, 127.7, 127.5, 127.4, 93.6, 93.2, 79.7, 76.7, 75.4, 74.9, 73.8, 71.8, 71.4, 71.2, 67.3, 60.4, 58.3, 17.0, 16.9; HRMS (ESI): Calcd for C.sub.20H.sub.23N.sub.3O.sub.4Na.sup.+ [M+Na].sup.+ 392.1586, found 392.1583. According to general procedure (C), the lactol (75 mg, 0.20 mmol) was reacted with trichloroacetonitrile (0.20 mL, 2.0 mmol) and K.sub.2CO.sub.3 (48 mg, 0.35 mmol) in DCM (2 mL) to afford compound 8* (65 mg, 0.13 mmol, 62%). [].sub.D.sup.20=32.3 (c=1.5, CHCl.sub.3); IR v.sub.max (film) 3336, 2910, 2113, 1672, 1454, 1356, 1273, 1157, 1063 cm.sup.1; .sup.1H-NMR (400 MHz, CDCl.sub.3) 8.65-8.49 (m, 1H), 7.43-7.22 (m, 10H), 6.19 (d, J=1.5 Hz, 1H), 5.03 (d, J=11.7 Hz, 1H), 4.78 (d, J=11.8 Hz, 1H), 4.70 (d, J=3.7 Hz, 2H), 4.09-3.98 (m, 3H), 3.69 (dd, J=1.6, 1.2 Hz, 1H), 1.23 (d, J=6.5 Hz, 3H); .sup.13C-NMR (100 MHz, CDCl.sub.3) 159.9, 138.2, 137.2, 128.9, 128.8 (2C), 128.7, 128.4, 128.3, 128.2, 128.0, 127.9 (2C), 97.0, 91.0, 76.0, 75.1, 74.5, 71.2, 70.3, 56.2, 16.9; HRMS (MALDI-TOF): Calcd for C.sub.22H.sub.23Cl.sub.3N.sub.4O.sub.4Na.sup.+ [M+Na].sup.+ 535.0677, found 535.0660.

Example 9: Synthesis of ethyl 2-O-benzoyl-3,4-di-O-benzyl-6-O-(2-naphthalenylmethyl)-1-thio--D-glucopyranoside (9*)

(100) ##STR00068##

(101) To a solution of 2-O-benzoyl-3-O-benzyl-4,6-O-benzylidene-1-thio--D-glucopyranoside (584 mg, 1.15 mmol) in DCM (10 mL), BH.sub.3-THF (1 M in THF, 6.9 mL, 6.9 mmol) and TMSOTf (0.1 mL, 0.6 mmol) were added dropwise at 0 C. The reaction was warmed to room temperature over 2 h, cooled to 0 C. again and quenched by drop wise addition of sat. aq. NaHCO.sub.3 solution. The emulsion was diluted with DCM and washed with a sat. aq. NaHCO.sub.3 solution. The organic phase was then dried over MgSO.sub.4, filtered and concentrated to give the crude alcohol. To a solution of crude alcohol in THF/DMF (9:1, 10 mL) at 0 C., naphtyl bromide (509 mg, 2.30 mmol) and NaH (33 mg, 1.38 mmol) were added. The reaction was warmed to room temperature and stirred for 30 min, cooled to 0 C. and quenched by the addition of water. After dilution with Et.sub.2O the organic phase was washed with 0.1 M HCl and sat. aq. NaHCO.sub.3 solutions. The organic phase was then dried over MgSO.sub.4, filtered and concentrated. Column chromatography on silica gel (hexanes/ethyl acetate) afforded 9* (626 mg, 0.97 mmol, 84%). [].sub.D.sup.20=+29.5 (c=3.1, CHCl.sub.3), IR v.sub.max (film) 3061, 3030, 2867, 1722, 1602, 1496, 1452, 1359, 1315, 1264, 1087, 1067, 1026 cm.sup.1; .sup.1H-NMR (400 MHz, CDCl.sub.3) 8.06-8.01 (m, 2H), 7.86-7.79 (m, 4H), 7.60-7.42 (m, 6H), 7.26-7.04 (m, 10H), 5.34 (dd, J=10.0, 8.9 Hz, 1H), 4.84-4.54 (m, 7H), 3.88-3.74 (m, 4H), 3.60 (ddd, J=9.5, 4.5, 2.0 Hz, 1H), 2.81-2.68 (m, 2H), 1.26 (t, J=7.5 Hz, 3H); .sup.13C-NMR (100 MHz, CDCl.sub.3) 165.4, 138.0, 137.9, 135.8, 133.4, 133.3, 133.2, 130.1, 130.0, 128.5 (2C), 128.4, 128.3, 128.1 (3C), 127.9, 127.8 (2C), 126.6, 126.2, 126.0, 125.9, 84.5, 83.4, 79.7, 78.1, 75.4, 75.2, 73.7, 72.6, 69.1, 24.1, 15.1; HRMS (MALDI-TOF): Calcd for C.sub.40H.sub.40O.sub.6SNa.sup.+ [M+Na].sup.+ 671.2438, found 671.2478.

Example 10: Synthesis of ethyl 3,4-di-O-benzyl-2-O-levulinoyl-6-O-(2-naphthalenylmethyl)-1-thio--D-glucopyranoside (10*)

(102) ##STR00069##

(103) To a solution of 9* (367 mg, 0.57 mmol) in DCM (5.0 mL) was added 0.5 M NaOMe in MeOH (5.0 mL) and stirred for 16 h. The mixture was neutralized with Amberlite IR 120 (H.sup.+) ion exchange resin, filtered and concentrated. The residue was dissolved in DCM (5.0 mL) together with DMAP (7 mg, 0.06 mmol), levulinic acid (81 L, 0.79 mmol) and EDC (120 L, 0.68 mmol). The reaction mixture was stirred for 4 h, concentrated and redissolved in EtOAc. The organic layer was washed with sat. aq. NH.sub.4Cl, sat. aq. NaHCO.sub.3 and brine, then dried over MgSO.sub.4 and concentrated. Column chromatography on silica gel (hexanes/ethyl acetate) afforded 10* (271 mg, 0.42 mmol, 75%). [].sub.D.sup.20=1.0 (c=1.5, CHCl.sub.3), IR v.sub.max (film) 3061, 3031, 2926, 2869, 1746, 1718, 1497, 1454, 1404, 1361, 1203, 1158, 1081, 1062 cm.sup.1; .sup.1H-NMR (400 MHz, CDCl.sub.3) 7.89-7.73 (m, 4H), 7.55-7.42 (m, 3H), 7.41-6.97 (m, 10H), 5.11-5.02 (m, 1H), 4.82-4.69 (m, 5H), 4.56 (d, J=10.8 Hz, 1H), 4.39 (d, J=10.0 Hz, 1H), 3.82-3.68 (m, 4H), 3.53 (ddd, J=9.2, 4.6, 2.1 Hz, 1H), 2.80-2.64 (m, 4H), 2.60-2.46 (m, 2H), 2.16 (s, 3H), 1.28 (t, J=7.5 Hz, 3H); .sup.13C-NMR (100 MHz, CDCl.sub.3) 206.2, 171.6, 138.3, 137.9, 135.7, 133.3, 133.1, 128.4 (2C), 128.3, 128.2, 128.0 (2C), 127.9 (3C), 127.8 (2C), 126.5, 126.2, 125.9, 125.8, 84.4, 83.5, 79.5, 77.9, 75.2, 75.1, 73.6, 72.3, 68.9, 38.0, 29.9, 28.2, 24.0, 15.0; HRMS (MALDI-TOF): Calcd for C.sub.38H.sub.42O.sub.7SNa.sup.+ [M+Na].sup.+ 665.2543, found 665.2558.

Example 11: Synthesis of N-(benzyl)benzyloxycarbonyl-5-amino-pentanyl 2-azido-3-O-benzyl-2-deoxy-4-O-(2-naphthalenylmethyl)--D-fucopyranoside (11*)

(104) ##STR00070##

(105) To a solution of 6* (50 mg, 78 mol) in DMF (4 ml) at 0 C., BnBr (28 L, 240 mol), TBAI (3 mg, 8 mol) and NaH (3 mg, 125 mmol) were added. After 3 h the reaction was quenched with MeOH at 0 C. and diluted with Et.sub.2O. The organic layer was washed with 0.1 M aq. HCl solution, sat. aq. NaHCO.sub.3 and brine. The organic layers were dried over MgSO.sub.4 and concentrated. Column chromatography on silica gel (hexanes/ethyl acetate) afforded 11* (32 mg, 44 mol, 56%). [].sub.D.sup.20=24.2 (c=1.6, CHCl.sub.3); IR v.sub.max (film) 3031, 2935, 2110, 1697, 1496, 1454, 1421, 1362, 1230, 1172, 1111, 1068 cm.sup.1; .sup.1H-NMR (400 MHz, CDCl.sub.3) 7.42 (s, 22H), 5.19 (d, J=12.8 Hz, 2H), 5.09 (d, J=11.9 Hz, 1H), 4.86 (d, J=11.9 Hz, 1H), 4.79-4.68 (m, 2H), 4.51 (d, J=8.8 Hz, 2H), 4.22-4.11 (m, 1H), 3.96-3.80 (m, 2H), 3.58 (d, J=2.5 Hz, 1H), 3.51-3.36 (m, 2H), 3.34-3.17 (m, 3H), 1.67-1.47 (m, 4H), 1.46-1.28 (m, 2H), 1.21 (d, J=6.4 Hz, 3H); .sup.13C-NMR (100 MHz, CDCl.sub.3) 138.0, 137.9, 135.8, 133.2, 133.1, 128.6 (2C), 128.6, 128.1 (2C), 128.0, 127.9, 127.8, 127.3, 126.8, 126.1, 126.0, 102.4, 81.1, 74.8, 74.7, 72.9, 70.6, 69.8, 67.2, 63.3, 50.6, 50.3, 47.2, 46.3, 29.3, 28.0, 27.6, 23.3, 17.1; HRMS (MALDI-TOF): Calcd for C.sub.44H.sub.48N.sub.4O.sub.6Na.sup.+ [M+Na].sup.+ 751.3466, found 751.3417.

Example 12: Synthesis of N-(benzyl)benzyloxycarbonyl-5-amino-pentanyl 2-N-acetyl-3-O-benzyl-2-deoxy-4-O-(2-naphthalenylmethyl)--D-fucosaminopyranoside (12*)

(106) ##STR00071##

(107) According to general procedure (F), azido-monosaccharide 11* (32 mg, 44 mol) was reacted with thioacetic acid for 24 h to give 12* (26 mg, 35 mol, 80%). [].sub.D.sup.20=+8.8 (c=1.4, CHCl.sub.3); IR v.sub.max (film) 3288, 3062, 2933, 1698, 1654, 1555, 1496, 1454, 1422, 1366, 1308, 1231, 1173, 1113, 1068 cm.sup.1; .sup.1H-NMR (400 MHz, CDCl.sub.3) 7.93-7.08 (m, 22H), 5.17 (d, J=15.1 Hz, 2H), 5.06 (d, J=11.9 Hz, 1H), 4.95 (d, J=8.3 Hz, 1H), 4.84 (d, J=12.0 Hz, 1H), 4.72-4.63 (m, 1H), 4.60-4.32 (m, 4H), 3.92-3.72 (m, 1H), 3.65 (d, J=2.2 Hz, 1H), 3.57 (q, J=6.3 Hz, 1H), 3.53-3.32 (m, 2H), 3.32-3.09 (m, 2H), 1.98-1.79 (m, 3H), 1.63-1.41 (m, 4H), 1.33-1.16 (m, 5H); .sup.13C-NMR (100 MHz, CDCl.sub.3) 171.1, 138.0, 133.2, 133.0, 128.6, 128.1, 128.0 (2C), 127.9, 127.8, 127.3, 127.0, 126.7, 126.1, 125.9, 99.4, 78.2, 75.5, 74.7, 72.6, 70.4, 69.5, 67.2, 55.5, 50.3, 47.3, 29.1, 27.5, 23.8, 23.5, 17.3; HRMS (MALDI-TOF): Calcd for C.sub.46H.sub.52N.sub.2O.sub.7Na.sup.+ [M+Na].sup.+ 767.3667, found 767.3575.

Example 13: Synthesis of N-(benzyl)benzyloxycarbonyl-5-amino-pentanyl 2-N-acetyl-3-O-benzyl-2-deoxy--D-fucosaminopyranoside (13*)

(108) ##STR00072##

(109) According to general procedure (G), monosaccharide 12* (20 mg, 27 mol) was reacted with DDQ (12 mg, 53 mol) in DCM/H.sub.2O to give alcohol 13* (12 mg, 20 mol, 74%). [].sub.D.sup.20=+13.9 (c=1.2, CHCl.sub.3); IR v.sub.max (film) 3298, 3032, 2934, 1697, 1657, 1554, 1497, 1454, 1423, 1369, 1303, 1229, 1173, 1069 cm.sup.1; .sup.1H-NMR (400 MHz, CDCl.sub.3) 7.53-7.07 (m, 15H), 5.16 (d, J=18.4 Hz, 2H), 4.85 (d, J=8.4 Hz, 1H), 4.67 (d, J=11.7 Hz, 1H), 4.59-4.42 (m, 3H), 4.31-4.15 (m, 1H), 3.92-3.72 (m, 2H), 3.61 (q, J=6.4 Hz, 1H), 3.45-3.10 (m, 4H), 2.34 (s, 1H), 1.99-1.80 (m, 3H), 1.64-1.43 (m, 4H), 1.36-1.20 (m, 5H); .sup.13C-NMR (100 MHz, CDCl.sub.3) 170.9, 138.0, 128.7, 128.6, 128.1 (2C), 127.9, 127.4, 127.3, 99.4, 77.4, 76.7, 71.9, 70.0, 69.5, 68.7, 67.3, 54.6, 50.5, 50.3, 47.4, 29.3, 29.0, 27.5, 23.8, 23.6, 16.7; HRMS (MALDI-TOF): Calcd for C.sub.35H.sub.77N.sub.2O.sub.7Na.sup.+ [M+Na].sup.+ 627.3041, found 627.2969.

Example 14: Synthesis of 5-amino-pentanyl 2-acetamido-2,5-dideoxy--D-xylo-hexos-4-uloside (14*)

(110) ##STR00073##

(111) To a solution of alcohol 13* (12 mg, 20 mol) in DCM (2 mL) Dess Martin periodinane (17 mg, 40 mol) was added and stirred for 12 h. The reaction mixture was loaded onto a silica gel column and purified by column chromatography (hexanes/acetone) to give the crude protected ketone. According to general procedure (I), the crude protected ketone was subjected to hydrogenolysis to give 14* as a minor component in a mixture of compounds. HRMS (ESI): Calcd for C.sub.13H.sub.26N.sub.2O.sub.6Na.sup.+ [M+H.sub.2O+Na].sup.+ 329.1689, found 329.1479.

Example 15: Synthesis of 5-amino-pentanyl 2-N-acetyl-2-deoxy--D-fucosaminopyranoside (15*)

(112) ##STR00074##

(113) According to general procedure (I), D-fucosaminoside 12* (7 mg, 9 mol) was subjected to hydrogenolysis to give 15* (2.5 mg, 8.6 mol, 92%). .sup.1H-NMR (600 MHz, D.sub.2O) 4.43 (d, J=8.5 Hz, 1H), 3.94-3.81 (m, 2H), 3.80-3.68 (m, 3H), 3.64-3.56 (m, 1H), 3.06-2.96 (m, 2H), 2.05 (s, 3H), 1.69 (dt, J=15.2, 7.6 Hz, 2H), 1.63-1.57 (m, 2H), 1.45-1.37 (m, 2H), 1.28 (d, J=6.3 Hz, 3H); .sup.13C-NMR (150 MHz, D.sub.2O) 177.2, 104.1, 73.8, 73.5, 73.1, 72.6, 54.8, 41.9, 30.8, 29.0, 24.8, 24.7, 18.1; HRMS (ESI): Calcd for C.sub.13H.sub.27N.sub.2O.sub.5.sup.+ [M+H].sup.+ 291.1920, found 291.1925.

Example 16: Synthesis of N-(benzyl)benzyloxycarbonyl-5-amino-pentanyl 2-azido-2-deoxy--L-fucopyranosyl-(13)-2-azido-2-deoxy-4-O-(2-naphthalenylmethyl)--D-fucopyranoside (16*)

(114) ##STR00075##

(115) According to general procedure (E), 2-azido-3,4-di-O-benzyl-2-deoxy--L-fucopyranosyl trichloroacetimidate (140 mg, 335 mol) and 6* (100 mg, 157 mol) were reacted in DCM (2 mL) at 20 C. to 15 C. over 30 min to give the crude disaccharide. To a solution of the crude disaccharide in DCM/MeOH (1:1, 2 mL) was added 0.5 M NaOMe in MeOH (0.1 mL) and stirred for 16 h. The mixture was neutralized with Amberlite IR 120 (H.sup.+) ion exchange resin, filtered and concentrated. Column chromatography (DCM/MeOH/acetone) afforded disaccharide 16* (110 mg, 136 mol, 87%). [].sub.D.sup.20=41.6 (c=1.5, CHCl.sub.3); IR v.sub.max (film) 3328, 2936, 2119, 1697, 1422, 1255, 1095, 1070, 1028 cm.sup.1; .sup.1H-NMR (400 MHz, CDCl.sub.3) 7.94-7.72 (m, 4H), 7.59-7.44 (m, 3H), 7.41-7.03 (m, 10H), 5.24 (d, J=3.6 Hz, 1H), 5.17 (d, J=12.9 Hz, 2H), 4.99 (d, J=12.2 Hz, 1H), 4.80 (d, J=12.2 Hz, 1H), 4.49 (d, J=6.9 Hz, 2H), 4.21 (t, J=9.3 Hz, 1H), 3.99-3.84 (m, 2H), 3.59-3.39 (m, 6H), 3.31-3.12 (m, 4H), 2.30 (s, 2H), 1.69-1.47 (m, 4H), 1.43-1.27 (m, 5H), 1.10 (d, J=6.6 Hz, 3H); .sup.13C-NMR (100 MHz, CDCl.sub.3) 136.0, 133.2, 133.1, 128.6, 128.3, 128.0, 127.9, 127.8, 127.4, 126.8, 126.6, 126.5, 126.3, 102.8, 99.9, 79.1, 78.2, 75.6, 71.4, 71.0, 69.9, 67.9, 67.3, 66.3, 63.8, 59.7, 50.6, 50.3, 47.2, 46.3, 29.3, 23.3, 17.3, 16.2; HRMS (MALDI-TOF): Calcd for C.sub.43H.sub.51N.sub.7O.sub.9Na.sup.+ [M+Na].sup.+ 832.3640, found 832.3676.

Example 17: Synthesis of N-(benzyl)benzyloxycarbonyl-5-amino-pentanyl 3,4-di-O-benzyl-2-azido-2-deoxy--L-fucopyranosyl-(13)-2-azido-2-deoxy-4-O-(2-naphthalenylmethyl)--D-fucopyranoside (17*)

(116) ##STR00076##

(117) To a solution of diol 16* (87 mg, 107 mol) in DMF (5 mL) at 0 C., BnBr (77 L, 645 mol), TBAI (4 mg, 11 mol) and NaH (8 mg, 333 mmol) were added. After 3 h the reaction was quenched with MeOH at 0 C. and diluted with Et.sub.2O. The organic layer was washed with 0.1 M aq. HCl solution, sat. aq. NaHCO.sub.3 and brine. The organic layers were dried over MgSO.sub.4 and concentrated. Column chromatography on silica gel (hexanes/ethyl acetate) afforded 17* (88 mg, 89 mol, 83%). [].sub.D.sup.20=50.2 (c=1.4, CHCl.sub.3); IR v.sub.max (film) 2937, 2114, 1697, 1496, 1454, 1421, 1360, 1233, 1171, 1103, 1068, 1038 cm.sup.1; .sup.1H-NMR (400 MHz, CDCl.sub.3) 7.92-7.72 (m, 4H), 7.63-7.07 (m, 23H), 5.22 (d, J=3.7 Hz, 1H), 5.18 (d, J=13.1 Hz, 2H), 4.95 (d, J=12.1 Hz, 1H), 4.83 (dd, J=12.5 Hz, 2H), 4.57-4.36 (m, 5H), 4.21 (dd, J=11.6, 8.1 Hz, 1H), 4.02-3.84 (m, 2H), 3.75 (dd, J=10.8, 3.5 Hz, 1H), 3.64-3.40 (m, 6H), 3.33-3.15 (m, 3H), 1.70-1.49 (m, 4H), 1.43-1.26 (m, 5H), 1.00 (d, J=6.5 Hz, 3H); .sup.13C-NMR (100 MHz, CDCl.sub.3) 138.1, 137.6, 136.0, 133.2, 133.0, 128.6, 128.5, 128.4, 128.3, 128.0, 127.9 (2C), 127.8, 127.3, 126.5, 126.4, 126.2, 102.7, 100.4, 79.4, 78.9, 75.8, 75.7, 74.9, 72.2, 70.9, 70.0, 67.2, 63.9, 59.4, 50.6, 50.3, 47.2, 46.3, 29.3, 28.0, 27.5, 23.3, 17.3, 16.8; HRMS (MALDI-TOF): Calcd for C.sub.57H.sub.63N.sub.7O.sub.9Na.sup.+ [M+Na].sup.+ 1012.4579, found 1012.4502.

Example 18: Synthesis of N-(benzyl)benzyloxycarbonyl-5-amino-pentanyl 2-N-acetyl-3,4-di-O-benzyl--L-fucosaminopyranosyl-(13)-2-N-acetyl-4-O-(2-naphthalenylmethyl)--D-fucosaminopyranoside (18*)

(118) ##STR00077##

(119) According to general procedure (F), azido-disaccharide 17* (88 mg, 89 mol) was reacted with thioacetic acid for 24 h to give 18* (82 mg, 80 mol, 90%). [].sub.D.sup.20=21.0 (c=1.2, CHCl.sub.3); IR v.sub.max (film) 3314, 2933, 1682, 1497, 1454, 1366, 1304, 1233, 1172, 1104, 1063, 1029 cm.sup.1; .sup.1H-NMR (400 MHz, CDCl.sub.3) 7.91-7.71 (m, 4H), 7.55-7.04 (m, 23H), 5.21-5.08 (m, 2H), 5.00-4.62 (m, 5H), 4.60-4.40 (m, 4H), 4.35-4.00 (m, 3H), 3.90-3.66 (m, 3H), 3.57-3.09 (m, 7H), 2.07-1.84 (m, 6H), 1.65-1.40 (m, 4H), 1.36-1.15 (m, 5H), 1.05 (d, J=6.5 Hz, 3H); .sup.13C-NMR (100 MHz, CDCl.sub.3) 171.4, 170.8, 133.2, 132.9, 128.6, 128.5, 128.4 (2C), 128.3, 128.2, 128.0, 127.9, 127.8, 127.6, 127.5, 127.4, 127.3, 126.4, 126.1, 126.0, 125.5, 101.4, 100.5, 78.5, 75.5, 74.3, 72.2, 70.7, 68.5, 67.6, 67.2, 52.0, 50.5, 48.1, 47.6, 29.2, 28.7, 23.6, 23.5, 17.4, 17.0; HRMS (MALDI-TOF): Calcd for C.sub.61H.sub.71N.sub.3O.sub.11Na.sup.+ [M+Na].sup.+ 1044.4981, found 1044.4917.

Example 19: Synthesis of N-(benzyl)benzyloxycarbonyl-5-amino-pentanyl 2-N-acetyl-3,4-di-O-benzyl--L-fucosaminopyranosyl-(13)-2-N-acetyl--D-fucosaminopyranoside (19*)

(120) ##STR00078##

(121) According to general procedure (G), disaccharide 18* (46 mg, 45 mol) was reacted with DDQ (30 mg, 132 mol) in DCM/H.sub.2O to give alcohol 19* (30 mg, 34 mol, 76%). [].sub.D.sup.20=35.7 (c=1.0, CHCl.sub.3); IR v.sub.max (film) 3290, 3088, 2932, 1702, 1646, 1548, 1497, 1454, 1422, 1372, 1304, 1227, 1101, 1063, 1024 cm.sup.1; .sup.1H-NMR (400 MHz, CDCl.sub.3) 7.41-7.14 (m, 20H), 5.22-5.07 (m, 2H), 5.01-4.85 (m, 2H), 4.84-4.31 (m, 6H), 4.29-4.18 (m, 1H), 4.11-3.89 (m, 2H), 3.87-3.38 (m, 6H), 3.36-3.04 (m, 3H), 2.00 (s, 3H), 1.91 (s, 3H), 1.63-1.38 (m, 4H), 1.27 (d, J=6.4 Hz, 5H), 1.17 (d, J=6.5 Hz, 3H); .sup.13C-NMR (100 MHz, CDCl.sub.3) 170.8, 128.7, 128.6, 128.5, 128.4 (2C), 128.0, 127.6, 127.5, 127.4, 101.5, 101.1, 80.4, 78.4, 75.8, 74.8, 72.0, 71.5, 70.1, 68.8, 67.4, 67.3, 51.3, 50.6, 48.8, 47.6, 28.7, 27.0, 23.6, 23.5, 17.2, 16.5; HRMS (MALDI-TOF): Calcd for C.sub.50H.sub.63N.sub.3O.sub.11Na.sup.+ [M+Na].sup.+ 904.4355, found 904.4344.

Example 20: Synthesis of 5-amino-pentanyl 2-N-acetyl--L-fucosaminopyranosyl-(13)-2-acetamido-2,5-dideoxy--D-xylo-hexos-4-uloside (20*)

(122) ##STR00079##

(123) To a solution of 19* (9 mg, 10 mol) in DCM (2 mL) Dess Martin periodinane (9 mg, 9 mol) was added and stirred for 12 h. The reaction mixture was loaded onto a silica gel column and purified by column chromatography (hexanes/acetone) to give the crude protected ketone. According to general procedure (I), the crude protected ketone was subjected to hydrogenolysis to give 20* as a mixture of compounds (approx. 2 mg, approx. 4 mol, approx. 39%). .sup.1H-NMR (600 MHz, D.sub.2O) 5.02 (d, J=4.0 Hz, 1H), 4.49 (q, J=6.5 Hz, 1H), 4.45 (d, J=8.8 Hz, 1H), 4.15 (dd, J=11.2, 4.1 Hz, 1H), 3.99 (dd, J=11.2, 3.2 Hz, 1H), 3.92-3.83 (m, 3H), 3.66-3.54 (m, 3H), 3.01-2.97 (m, 2H), 2.06 (s, 3H), 1.98 (s, 3H), 1.70-1.64 (m, 2H), 1.61-1.55 (m, 2H), 1.42-1.36 (m, 2H), 1.29 (d, J=6.4 Hz, 3H), 1.20 (d, J=6.6 Hz, 3H); .sup.13C-NMR (150 MHz, D.sub.2O) 177.0, 176.4, 104.0, 100.9, 95.4, 81.4, 75.9, 73.7, 72.8, 70.2, 69.9, 52.1, 42.0, 30.8, 29.1, 24.9, 24.8, 24.7, 17.9, 13.8; HRMS (ESI): Calcd for C.sub.21H.sub.40N.sub.3O.sub.10.sup.+ [M+H.sub.2O+H].sup.+ 494.2714, found 494.2740.

Example 21: Synthesis of 5-amino-pentanyl 2-N-acetyl--L-fucosaminopyranosyl-(13)-2-N-acetyl--D-fucosaminopyranoside (21*)

(124) ##STR00080##

(125) According to general procedure (I), disaccharide 20* (7 mg, 7 mol) was subjected to hydrogenolysis to give 21* (2.73 mg, 5.7 mol, 83%). .sup.1H-NMR (600 MHz, D.sub.2O) 5.00 (d, J=4.0 Hz, 1H), 4.41 (d, J=8.5 Hz, 1H), 4.15-4.09 (m, 2H), 4.01-3.94 (m, 2H), 3.89 (dt, J=10.2, 6.0 Hz, 1H), 3.85 (d, J=3.0 Hz, 1H), 3.82-3.74 (m, 3H), 3.58 (dt, J=10.1, 6.4 Hz, 1H), 3.04-2.96 (m, 2H), 2.05 (s, 3H), 2.00 (s, 3H), 1.72-1.64 (m, 2H), 1.63-1.55 (m, 2H), 1.44-1.36 (m, 2H), 1.28 (d, J=6.5 Hz, 3H), 1.24 (d, J=6.6 Hz, 3H); .sup.13C-NMR (150 MHz, D.sub.2O) 176.8, 176.7, 104.2, 101.6, 79.4, 73.6, 73.3, 73.1, 72.7, 70.1, 69.7, 54.0, 52.1, 41.9, 30.8, 29.1, 24.8 (2C), 24.7, 18.0 (2C); HRMS (ESI): Calcd for C.sub.21H.sub.40N.sub.3O.sub.9.sup.+ [M+H].sup.+ 478.2765, found 478.2775.

Example 22: Synthesis of N-(benzyl)benzyloxycarbonyl-5-amino-pentanyl 3,4-di-O-benzyl-2-O-levulinoyl-6-O-(2-naphthalenylmethyl)--D-glucopyranoside (22*)

(126) ##STR00081##

(127) According to general procedure (B), thioglucoside 10* (30 mg, 47 mol) was reacted with N-(benzyl)benzyloxycarbonyl-5-amino-pentanol (31 mg, 93 mol), NIS (13 mg, 56 mol) and TfOH (0.8 L, 9 mol) in DCM (1 mL) at 30 C. to 20 C. over 1 h. After work-up column chromatography (hexanes/EtOAc) afforded 22* (26 mg, 29 mol, 61%). [].sub.D.sup.20=+3.7 (c=0.9, CHCl.sub.3); IR v.sub.max (film) 2924, 1747, 1698, 1454, 1421, 1362, 1210, 1153, 1071 cm.sup.1; .sup.1H-NMR (400 MHz, CDCl.sub.3) 7.84-7.76 (m, 4H), 7.26 (s, 22H), 7.09-7.05 (m, 2H), 5.21-5.13 (m, 3H), 4.98 (t, J=8.6 Hz, 1H), 4.77 (d, J=10.9 Hz, 3H), 4.70 (d, J=11.6 Hz, 2H), 4.53-4.44 (m, 4H), 4.37-4.27 (m, 1H), 3.85-3.59 (m, 5H), 3.53-3.11 (m, 3H), 2.82-2.39 (m, 4H), 2.17-2.03 (m, 2H), 1.62-1.43 (m, 8H), 1.37-1.17 (m, 3H); .sup.13C-NMR (100 MHz, CDCl.sub.3) 171.5, 138.4, 133.4, 133.1, 128.7, 128.5, 128.3, 128.1 (2C), 128.0, 127.9, 127.8 (2C), 126.7, 126.2, 126.0, 101.1, 83.0, 78.1, 75.2 (2C), 75.1, 73.7 (2C), 69.6, 68.8, 67.3, 50.3, 47.2, 37.9, 30.0, 29.3, 28.1, 23.3; HRMS (MALDI-TOF): Calcd for C.sub.56H.sub.61NO.sub.10Na.sup.+ [M+Na].sup.+ 930.4188, found 930.4190.

Example 23: Synthesis of N-(benzyl)benzyloxycarbonyl-5-amino-pentanyl 3,4-di-O-benzyl-6-O-(2-naphthalenymethyl)--D-glucopyranoside (23*)

(128) ##STR00082##

(129) To a solution of 22* (13 mg, 14 mol) in DCM (1 mL) hydrazine hydrate (2.8 L, 57 mol) dissolved in AcOH (40 L) and pyridine (60 L) was added and the solution stirred for 1 h. The reaction was then quenched by the addition of acetone and concentrated. Column chromatography on silica gel (hexanes/ethyl acetate) afforded 23* (10 mg, 12 mol, 86%). [].sub.D.sup.20=+4.9 (c=1.0, CHCl.sub.3); IR v.sub.max (film) 2926, 1698, 1454, 1230, 1063 cm.sup.1; .sup.1H-NMR (400 MHz, CDCl.sub.3) 7.84-7.01 (m, 27H), 5.16 (d, J=9.8 Hz, 2H), 4.93 (t, J=11.1 Hz, 1H), 4.81 (dd, J=11.0, 6.1 Hz, 2H), 4.75 (d, J=12.4 Hz, 1H), 4.67 (d, J=12.4 Hz, 1H), 4.51-4.43 (m, 3H), 4.26-4.17 (m, 1H), 4.02-3.79 (m, 2H), 3.78-3.67 (m, 2H), 3.60-3.44 (m, 4H), 3.30-3.12 (m, 2H), 1.66-1.43 (m, 4H), 1.38-1.19 (m, 2H); .sup.13C-NMR (100 MHz, CDCl.sub.3) 138.0, 135.7, 133.4, 133.1, 128.7, 128.6, 128.5, 128.3, 128.1, 128.0 (2C), 127.8, 127.3, 126.8, 126.2, 126.0, 103.1, 84.7, 77.7, 75.3, 75.2, 74.8, 73.7, 70.1, 69.9, 69.0, 67.3, 50.3, 47.2, 46.2, 32.4, 29.3, 27.5, 23.5; HRMS (MALDI-TOF): Calcd for C.sub.51H.sub.55NO.sub.8Na.sup.+ [M+Na].sup.+ 832.3820, found 832.3870.

Example 24: Synthesis of N-(benzyl)benzyloxycarbonyl-5-amino-pentanyl 2-azido-3,4-di-O-benzyl-2-deoxy--L-pneumopyranosyl-(12)-3,4-di-O-benzyl-6-O-(2-naphthalenylmethyl)--D-glucopyranoside (24*)

(130) ##STR00083##

(131) According to general procedure (E), pneumosyl-imidate 8* (12 mg, 23 mol) and 23* (10 mg, 12 mol) were reacted in DCM (1 mL) at 30 C. to 20 C. over 30 min to give 24* (13 mg, 11 mol, 91%). [].sub.D.sup.20=0.7 (c=0.7, CHCl.sub.3); IR v.sub.max (film) 2929, 2868, 2112, 1697, 1496, 1454, 1421, 1361, 1229, 1126, 1057 cm.sup.1; .sup.1H-NMR (400 MHz, CDCl.sub.3) 7.86-7.72 (m, 4H), 7.50-7.43 (m, 3H), 7.41-7.02 (m, 30H), 5.24-5.08 (m, 3H), 4.93 (dd, J=30.9, 11.5 Hz, 2H), 4.77-4.44 (m, 10H), 4.26-4.08 (m, 2H), 3.85-3.67 (m, 4H), 3.66-3.51 (m, 5H), 3.47-3.29 (m, 2H), 3.25-3.09 (m, 2H), 1.61-1.38 (m, 4H), 1.32-1.03 (m, 5H); .sup.13C-NMR (100 MHz, CDCl.sub.3) 137.9, 133.4, 133.1, 128.8, 128.7 (2C), 128.6 (2C), 128.4, 128.2, 128.1, 128.0, 127.9, 127.8, 127.2, 126.8, 126.3, 126.0 (2C), 101.8, 99.2, 78.7, 77.4, 75.7, 75.1 (2C), 73.8 (2C), 70.9, 68.9, 67.3, 66.9, 29.5, 23.4, 17.0; HRMS (MALDI-TOF): Calcd for C.sub.71H.sub.76N.sub.4O.sub.11Na.sup.+ [M+Na].sup.+ 1183.5403, found 1183.5391.

Example 25: Synthesis of N-(benzyl)benzyloxycarbonyl-5-amino-pentanyl 2-N-acetyl-3,4-di-O-benzyl--L-pneumosaminopyranosyl-(12)-3,4-di-O-benzyl-6-O-(2-naphthalenylmethyl)--D-glucopyranoside (25*)

(132) ##STR00084##

(133) According to general procedure (F), azido-disaccharide 24* (13 mg, 11 mol) was reacted with thioacetic acid for 24 h to give 25* (10 mg, 8 mol, 76%). [].sub.D.sup.20=31.1 (c=1.0, CHCl.sub.3); IR v.sub.max (film) 2930, 1698, 1497, 1454, 1363, 1229, 1056 cm.sup.1; .sup.1H-NMR (400 MHz, CDCl.sub.3) 7.85-7.73 (m, 4H), 7.49-7.43 (m, 3H), 7.35-7.12 (m, 28H), 7.08-7.02 (m, 2H), 7.01-6.94 (m, 1H), 5.28 (s, 1H), 5.21-5.13 (m, 2H), 4.90-4.81 (m, 3H), 4.77-4.67 (m, 5H), 4.60-4.54 (m, 1H), 4.51-4.30 (m, 5H), 4.28-4.20 (m, 1H), 3.90-3.78 (m, 2H), 3.76-3.57 (m, 6H), 3.49-3.43 (m, 1H), 3.38 (s, 1H), 3.28-3.14 (m, 2H), 1.65 (s, 3H), 1.58-1.45 (m, 4H), 1.30-1.16 (m, 5H); .sup.13C-NMR (100 MHz, CDCl.sub.3) 169.8, 138.5, 138.3, 137.9, 135.7, 133.4, 133.2, 128.7 (2C), 128.5 (3C), 128.3 (2C), 128.2, 128.1, 128.0 (3C), 127.9, 127.7, 127.6, 126.8, 126.3, 126.0, 102.0, 100.3, 86.0, 79.0, 78.5, 77.4, 75.6 (2C), 75.1, 75.0, 73.8, 73.3, 70.0, 69.0, 67.3, 66.6, 50.7, 50.4, 47.6, 29.6, 27.7, 23.5, 23.3, 16.8; HRMS (MALDI-TOF): Calcd for C.sub.73H.sub.80N.sub.2O.sub.12Na.sup.+ [M+Na].sup.+ 1199.5603, found 1199.5599.

Example 26: Synthesis of N-(benzyl)benzyloxycarbonyl-5-amino-pentanyl 2-N-acetyl-3,4-di-O-benzyl--L-pneumosaminopyranosyl-(12)-3,4-di-O-benzyl--D-glucopyranoside (26*)

(134) ##STR00085##

(135) According to general procedure (G), disaccharide 25* (10 mg, 8 mol) was reacted with DDQ (6 mg, 25 mol) for 4 h to give primary alcohol 26* (7 mg, 7 mol, 79%). [].sub.D.sup.20=34.5 (c=0.7, CHCl.sub.3); IR v.sub.max (film) 3404, 2925, 1697, 1497, 1454, 1421, 1362 1260, 1027 cm.sup.1; .sup.1H-NMR (400 MHz, CDCl.sub.3) 7.39-7.16 (m, 30H), 7.04-6.93 (m, 1H), 5.28 (s, 1H), 5.23-5.14 (m, 2H), 4.92-4.83 (m, 3H), 4.80 (d, J=11.0 Hz, 1H), 4.72 (d, J=12.2 Hz, 2H), 4.63-4.55 (m, 2H), 4.53-4.47 (m, 2H), 4.45-4.37 (m, 1H), 4.35-4.23 (m, 2H), 3.90-3.72 (m, 3H), 3.72-3.53 (m, 5H), 3.47-3.30 (m, 2H), 3.29-3.14 (m, 2H), 1.66 (s, 3H), 1.58-1.45 (m, 4H), 1.30-1.16 (m, 5H); .sup.13C-NMR (100 MHz, CDCl.sub.3) 169.8, 138.5, 138.2, 138.0, 128.7 (3C), 128.5 (2C), 128.3, 128.2 (2C), 128.1, 128.0, 127.7, 127.6, 102.0, 100.4, 85.7, 78.9, 78.1, 77.4, 75.8, 75.6, 75.1, 73.2, 70.0, 67.4, 66.7, 62.1, 50.5, 47.6, 47.2, 29.4, 28.1, 27.4, 23.3, 16.7; HRMS (MALDI-TOF): Calcd for C.sub.62H.sub.72N.sub.2O.sub.12Na.sup.+ [M+Na].sup.+ 1059.4977, found 1059.4938.

Example 27: Synthesis of 5-amino-pentanyl 2-N-acetyl--L-pneumosaminopyranosyl-(12)--D-glucopyranosyluronate (27*)

(136) ##STR00086##

(137) According to general procedure (H), alcohol 26* (6 mg, 6 mol) was reacted with TEMPO (0.2 mg, 2 mol) and BAIB (9 mg, 30 mol) to give the protected uronate disaccharide. According to general procedure (I), the uronate disaccharide was subjected to hydrogenolysis to give 27* (1.6 mg, 3.4 mol, 59%). .sup.1H-NMR (600 MHz, D.sub.2O) 5.15 (s, 1H), 4.55 (d, J=7.9 Hz, 1H), 4.33 (q, J=6.7 Hz, 1H), 4.23 (d, J=4.8 Hz, 1H), 4.09 (dd, J=4.8, 3.2 Hz, 1H), 3.92 (dt, J=10.0, 6.9 Hz, 1H), 3.85-3.81 (m, 1H), 3.74-3.65 (m, 3H), 3.55-3.50 (m, 1H), 3.43 (dd, J=9.0, 8.0 Hz, 1H), 3.00 (t, J=7.6 Hz, 2H), 2.06 (s, 3H), 1.73-1.66 (m, 4H), 1.49-1.42 (m, 2H), 1.26 (d, J=6.6 Hz, 3H); .sup.13C-NMR (150 MHz, D.sub.2O) 178.4, 176.74, 103.6, 102.6, 80.8, 78.8, 78.7, 74.3, 73.6, 72.7, 69.9, 66.5, 53.8, 42.0, 30.9, 28.9, 25.0, 24.7, 18.1; HRMS (ESI): Calcd for C.sub.19H.sub.34N.sub.2O.sub.11Na.sup.+ [M+Na].sup.+ 489.2060, found 489.2060.

(138) Compounds 27*a-27*e constitute further examples according to the present invention that can be obtained following the procedure described for compound 27:

(139) ##STR00087## ##STR00088##

Example 28: Synthesis of N-(benzyl)benzyloxycarbonyl-5-amino-pentanyl 4-O-acetyl-2-azido-2-deoxy--L-fucopyranosyl-(13)-2-azido-2-deoxy-4-O-(2-naphthalenylmethyl)--D-fucopyranoside (28*)

(140) ##STR00089##

(141) To a solution of the disaccharide 16* (110 mg, 136 mol) in DMF (1 mL) was added trimethyl orthoacetate (104 L, 815 mol) and p-TSA (4 mg, 21 mol) and the reaction mixture stirred for 30 min. Triethylamine (2 drops) was added and the solvent removed under vacuum using toluene as an azeotrope. The crude residue was taken up in 80% acetic acid (3 mL) and the reaction mixture stirred for 1 h. The solvent was removed under vacuum and azeotroped with toluene. Column chromatography on silica gel (hexanes/EtOAc) afforded 28* (92 mg, 108 mol, 80%). [].sub.D.sup.20=50.9 (c=1.3, CHCl.sub.3); IR v.sub.max (film) 3436, 2937, 2114, 1743, 1696, 1423, 1363, 1232, 1165, 1125, 1093, 1070, 1036 cm.sup.1; .sup.1H-NMR (400 MHz, CDCl.sub.3) 7.95-7.74 (m, 4H), 7.59-7.47 (m, 3H), 7.41-7.10 (m, 10H), 5.24 (d, J=3.6 Hz, 1H), 5.17 (d, J=13.3 Hz, 2H), 5.05 (d, J=12.4 Hz, 1H), 4.74 (d, J=12.4 Hz, 1H), 4.49 (d, J=7.6 Hz, 2H), 4.41 (d, J=2.2 Hz, 1H), 4.21 (dd, J=11.2, 8.3 Hz, 1H), 4.01-3.84 (m, 2H), 3.60-3.37 (m, 6H), 3.31-3.16 (m, 3H), 2.09 (s, 3H), 1.67-1.48 (m, 4H), 1.44-1.27 (m, 5H), 0.94 (d, J=6.6 Hz, 3H); .sup.13C-NMR (100 MHz, CDCl.sub.3) 171.3, 138.0, 136.0, 133.1 (2C), 128.6, 128.4, 127.9, 127.8, 127.3, 126.9, 126.8, 126.7, 126.5, 102.7, 99.8, 79.2, 78.1, 75.8, 72.7, 71.0, 70.0, 67.2, 66.5, 65.4, 63.9, 59.6, 50.6, 50.3, 47.2, 46.3, 29.3, 28.0, 27.5, 23.3, 20.8, 17.3, 16.1; HRMS (MALDI-TOF): Calcd for C.sub.45H.sub.53N.sub.7O.sub.10Na.sup.+ [M+Na].sup.+ 874.3746, found 874.3737.

Example 29: Synthesis of N-(benzyl)benzyloxycarbonyl-5-amino-pentanyl 3,4-di-O-benzyl-6-O-(2-naphthalenylmethyl)--D-glucopyranosyl-(13)-4-O-acetyl-2-azido-2-deoxy--L-fucopyranosyl-(13)-2-azido-2-deoxy-4-O-(2-naphthalenylmethyl)--D-fucopyranoside (29*)

(142) ##STR00090##

(143) According to general procedure (B), thioglucoside 10* (75 mg, 117 mol) was reacted with disaccharide 28* (50 mg, 59 mol), NIS (29 mg, 129 mol) and TfOH (1 L, 12 mol) in DCM (2 mL) at 20 C. to 10 C. over 1 h to give the crude trisaccharide. To a solution of the crude trisaccharide in DCM (3 mL) hydrazine hydrate (11 L, mol) dissolved in AcOH (40 L) and pyridine (60 L) was added and the solution stirred for 1 h. The reaction was then quenched by the addition of acetone and concentrated. Column chromatography on silica gel (hexanes/ethyl acetate) afforded alcohol 29* (72 mg, 54 mol, 91%). [].sub.D.sup.20=19.2 (c=1.0, CHCl.sub.3); IR v.sub.max (film) 2936, 2115, 1697, 1454, 1360, 1235, 1069 cm.sup.1; .sup.1H-NMR (400 MHz, CDCl.sub.3) 7.87-7.72 (m, 8H), 7.50-7.17 (m, 24H), 7.10-7.03 (m, 2H), 5.33 (d, J=3.6 Hz, 1H), 5.19 (d, J=12.8 Hz, 2H), 5.05 (d, J=11.3 Hz, 1H), 4.98 (d, J=12.3 Hz, 1H), 4.88-4.70 (m, 6H), 4.60-4.46 (m, 4H), 4.32-4.19 (m, 1H), 4.09 (d, J=7.0 Hz, 1H), 4.01-3.86 (m, 3H), 3.74-3.66 (m, 3H), 3.63-3.42 (m, 7H), 3.40 (d, J=2.1 Hz, 1H), 3.33-3.18 (m, 3H), 2.13 (s, 3H), 1.71-1.50 (m, 4H), 1.47-1.21 (m, 5H), 0.90 (d, J=6.5 Hz, 3H); .sup.13C-NMR (100 MHz, CDCl.sub.3) 174.1, 138.4, 138.2, 138.0, 137.9, 135.6, 133.2, 133.0, 132.9, 128.5, 128.4, 128.3, 128.1 (2C), 128.0, 127.9, 127.8, 127.7 (2C), 127.6 (2C), 127.5, 127.3, 127.2, 126.7, 126.5, 126.4, 126.3, 126.1, 126.0, 125.7, 102.7, 100.3, 100.2, 85.1, 82.0, 77.9, 77.4, 77.0, 76.7, 75.5, 75.5, 75.0, 74.9, 74.5, 74.2, 73.4, 70.8, 69.8, 68.5, 67.1, 63.8, 58.1, 29.2, 27.9, 23.2, 17.1, 16.6; HRMS (MALDI-TOF): Calcd for C.sub.76H.sub.83N.sub.7O.sub.15Na.sup.+ [M+Na].sup.+ 1356.5839, found 1356.5896.

Example 30: Synthesis of N-(benzyl)benzyloxycarbonyl-5-amino-pentanyl 2-azido-3,4-di-O-benzyl-2-deoxy--L-pneumopyranosyl-(12)-3,4-di-O-benzyl-6-O-(2-naphthalenylmethyl)--D-glucopyranosyl-(13)-4-O-acetyl-2-azido-2-deoxy--L-fucopyranosyl-(13)-2-azido-2-deoxy-4-O-(2-naphthalenyl methyl)--D-fucopyranoside (30*)

(144) ##STR00091##

(145) According to general procedure (E), pneumosyl-imidate 8* (100 mg, 196 mol) and trisaccharide 29* (58 mg, 43 mol) were reacted in DCM (2 mL) at 30 C. to 20 C. over 30 min to give tetrasaccharide 30* (40 mg, 24 mol, 55%). [].sub.D.sup.20=38.3 (c=1.2, CHCl.sub.3); IR v.sub.max (film) 2936, 2114, 1744, 1697, 1496, 1454, 1361, 1231, 1065 cm.sup.1; .sup.1H-NMR (400 MHz, CDCl.sub.3) 7.85-6.94 (m, 44H), 5.34 (d, J=3.7 Hz, 1H), 5.23 (s, 1H), 5.21-5.10 (m, 3H), 4.96 (dd, J=11.8, 3.2 Hz, 2H), 4.88 (d, J=11.3 Hz, 1H), 4.77-4.36 (m, 12H), 4.27-4.14 (m, 2H), 4.08-3.99 (m, 1H), 3.95-3.85 (m, 2H), 3.81-3.76 (m, 1H), 3.74-3.60 (m, 5H), 3.58-3.37 (m, 8H), 3.33-3.16 (m, 3H), 1.90 (s, 3H), 1.64-1.46 (m, 4H), 1.42-1.22 (m, 5H), 1.19 (d, J=6.5 Hz, 3H), 0.83 (d, J=6.4 Hz, 3H); .sup.13C-NMR (100 MHz, CDCl.sub.3) 170.3, 138.4, 138.0, 137.9, 137.7, 135.6, 133.2, 133.1, 133.0, 128.9, 128.7, 128.6 (2C), 128.5, 128.4, 128.3, 128.2, 128.1, 128.0 (2C), 127.9, 127.8, 127.7, 127.4, 127.2, 126.8, 126.5, 126.4, 126.1, 125.9, 125.8, 102.8, 100.1, 98.6, 96.7, 84.8, 78.7, 78.5, 77.4, 76.0, 75.3, 75.1, 75.0, 74.7, 73.3, 71.0, 70.9, 69.9, 68.9, 68.7, 67.3, 65.7, 63.9, 57.4, 57.0, 29.3, 27.6, 23.3, 20.9, 17.6, 16.8, 16.3; HRMS (MALDI-TOF): Calcd for C9.sub.6H.sub.104N.sub.10O.sub.18Na.sup.+ [M+Na].sup.+ 1707.7422, found 1707.7445.

Example 31: Synthesis of N-(benzyl)benzyloxycarbonyl-5-amino-pentanyl 2-N-acetyl-3,4-di-O-benzyl--L-pneumosaminopyranosyl-(12)-3,4-di-O-benzyl-6-O-(2-naphthalenylmethyl)--D-glucopyranosyl-(13)-4-O-acetyl-2-N-acetyl--L-fucosaminopyranosyl-(13)-2-N-acetyl-4-O-(2-naphthalenylmethyl)--D-fucosaminopyranoside (31*)

(146) ##STR00092##

(147) According to general procedure (F), azido-tetrasaccharide 30* (40 mg, 24 mol) was reacted with thioacetic acid for 48 h to give 31* (31 mg, 18 mol, 75%). [].sub.D.sup.20=58.3 (c=1.2, CHCl.sub.3); IR v.sub.max (film) 2963, 1740, 1674, 1519, 1454, 1365, 1260, 1234, 1025 cm.sup.1; .sup.1H-NMR (400 MHz, CDCl.sub.3) 7.89-6.61 (m, 44H), 5.40-5.04 (m, 6H), 4.95-4.80 (m, 2H), 4.79-4.11 (m, 16H), 4.10-3.92 (m, 2H), 3.91-3.73 (m, 4H), 3.74-3.54 (m, 4H), 3.54-3.31 (m, 4H), 3.30-3.02 (m, 4H), 2.00 (s, 3H), 1.90-1.85 (m, 3H), 1.68 (s, 3H), 1.58-1.38 (m, 4H), 1.38-1.23 (m, 5H), 1.20 (d, J=6.4 Hz, 3H), 1.10-1.00 (m, 3H); .sup.13C-NMR (100 MHz, CDCl.sub.3) 171.2, 170.7 (2C), 170.0, 138.5, 138.1, 137.9, 137.7, 136.2, 133.4, 133.1 (2C), 133.0, 128.8, 128.7, 128.5 (2C), 128.4, 128.2, 128.0, 127.9, 127.7 (2C), 127.6, 127.5, 127.4, 127.3, 126.7, 126.3, 125.9, 125.5, 100.7, 98.7, 98.6, 97.1, 84.1, 79.1, 78.4, 77.4, 75.6, 75.0, 74.5, 74.0, 73.8, 73.5, 73.3, 73.1, 70.7, 70.1, 69.7, 69.3, 68.1, 67.3, 66.9, 66.0, 50.4, 48.6, 47.6, 47.4, 29.0, 27.2, 23.8, 23.4, 23.1, 21.0, 17.3, 16.9, 16.7; HRMS (MALDI-TOF): Calcd for C.sub.102H.sub.116N.sub.4O.sub.21Na.sup.+ [M+Na].sup.+ 1755.8024, found 1755.8089.

Example 32: Synthesis of N-(Benzyl)benzyloxycarbonyl-5-amino-pentanyl 2-N-acetyl-3,4-di-O-benzyl-L-pneumosaminopyranosyl-(12)-3,4-di-O-benzyl--D-glucopyranosyl-(13)-2-N-acetyl--L-fucosaminopyranosyl-(13)-2-N-acetyl--D-fucosaminopyranoside (32*)

(148) ##STR00093##

(149) To a solution of 31* (19 mg, 11 mol) in DCM/MeOH (1:1, 1 mL) was added 0.5 M NaOMe in MeOH (0.5 mL) and stirred for 16 h. The mixture was neutralized with Amberlite IR 120 (H.sup.+) ion exchange resin, filtered and concentrated. According to general procedure (G), the crude material was reacted with DDQ (9 mg, 40 mol) in DCM/H.sub.2O to give triol 32* (10 mg, 7 mol, 64%). [].sub.D.sup.20=69.7 (c=1.0, CHCl.sub.3); IR v.sub.max (film) 3400, 3030, 2933, 1656, 1524, 1497, 1454, 1421, 1366, 1305, 1232, 1055 cm.sup.1; .sup.1H-NMR (400 MHz, CDCl.sub.3) 7.53-6.98 (m, 30H), 5.25-5.01 (m, 4H), 4.94-4.70 (m, 4H), 4.67-4.34 (m, 7H), 4.28-4.10 (m, 4H), 4.00-3.10 (m, 19H), 2.06 (s, 3H), 1.99-1.94 (m, 3H), 1.70 (s, 3H), 1.63-1.37 (m, 4H), 1.37-1.07 (m, 11H); 1.sup.3C-NMR (100 MHz, CDCl.sub.3) 171.6, 170.9, 170.1, 138.3, 128.7, 128.6 (2C), 128.5, 128.4, 128.3, 128.2, 128.1, 127.7, 127.5, 127.3, 127.2, 126.8, 101.7, 101.4, 101.2, 100.6, 99.5, 84.2, 80.8, 78.5, 77.5, 77.4, 77.2, 76.8, 75.7, 75.5, 75.2, 72.7, 72.5, 71.8, 71.3, 70.2, 69.7, 68.7, 68.6, 68.2, 67.3, 66.9, 61.9, 51.4, 50.6, 49.5, 48.5, 47.9, 23.9, 23.8, 23.5, 21.2, 16.6; HRMS (MALDI-TOF): Calcd for C.sub.78H.sub.98N.sub.4O.sub.20Na.sup.+ [M+Na].sup.+ 1433.6666, found 1433.6640.

Example 33: Synthesis of 5-amino-pentanyl 2-N-acetyl--L-pneumosaminopyranosyl-(12)--D-glucopyranosyl-(13)-2-N-acetyl--L-fucosaminopyranosyl-(13)-2-N-acetyl--D-fucosaminopyranoside (3*)

(150) ##STR00094##

(151) According to general procedure (H), triol 32* (10 mg, 7 mol) was reacted with TEMPO (0.3 mg, 2 mol) and BAIB (6 mg, 19 mol) to give the protected uronate tetrasaccharide. According to general procedure (I), the uronate tetrasaccharide was subjected to hydrogenolysis to give carboxylic acid 33* (4.9 mg, 5.8 mol, 83%). .sup.1H-NMR (600 MHz, D.sub.2O) 5.18 (s, 1H), 5.07 (d, J=3.9 Hz, 1H), 4.69 (d, J=7.8 Hz, 1H), 4.65 (q, J=6.7 Hz, 1H), 4.41 (d, J=8.6 Hz, 1H), 4.37 (dd, J=11.6, 3.0 Hz, 1H), 4.18 (d, J=4.8 Hz, 1H), 4.12 (dd, J=4.7, 3.2 Hz, 1H), 4.10-4.06 (m, 2H), 4.03 (dd, J=11.6, 3.9 Hz, 1H), 3.94 (t, J=9.6 Hz, 1H), 3.88 (dt, J=10.1, 6.0 Hz, 1H), 3.82-3.74 (m, 4H), 3.73-3.68 (m, 2H), 3.61-3.52 (m, 2H), 3.51 (dd, J=9.2, 7.9 Hz, 1H), 3.02-2.98 (m, 2H), 2.05 (s, 3H), 2.04 (s, 3H), 2.00 (s, 3H), 1.71-1.65 (m, 2H), 1.61-1.56 (m, 2H), 1.44-1.37 (m, 2H), 1.28 (d, J=6.5 Hz, 3H), 1.24 (d, J=6.6 Hz, 3H), 1.15 (d, J=6.6 Hz, 3H); .sup.13C-NMR (150 MHz, D.sub.2O) 178.4, 176.8, 176.5, 176.3, 104.2, 102.1, 101.0, 98.2, 79.1, 78.8, 78.1, 74.3, 73.7, 73.5, 73.2, 73.1, 72.6, 69.8, 69.6, 68.9, 66.4, 54.0, 53.7, 51.0, 41.9, 30.8, 29.0, 25.0, 24.9, 24.8, 24.7, 18.1, 18.0, 17.9; HRMS (ESI): Calcd for C.sub.35H.sub.60N.sub.4O.sub.19Na.sup.+ [M+Na].sup.+ 863.3744, found 864.3774.

(152) Compounds 33*a-33*e constitute further examples according to the present invention that can be obtained following the procedure described for compound 33*:

(153) ##STR00095## ##STR00096##

Example 34: Synthesis of N-(Benzyl)benzyloxycarbonyl-5-amino-pentanyl 2-azido-3,4-di-O-benzyl-2-deoxy-L-pneumopyranosides (34* and 34*)

(154) ##STR00097##

(155) According to general procedure (E), pneumosyl-imidate 8* (30 mg, 58 mol) and N-(benzyl)benzyloxycarbonyl-5-amino-pentanol (38 mg, 117 mol) were reacted in DCM (1 mL) at 30 C. to 20 C. over 30 min to give and anomers 34* (28 mg, 41 mol, 71%) and 34* (10 mg, 15 mol, 25%). 34*: [].sub.D.sup.20=18.0 (c=1.3, CHCl.sub.3); IR v.sub.max (film) 2934, 2111, 1697, 1496, 1454, 1422, 1360, 1229, 1061 cm.sup.1; .sup.1H-NMR (400 MHz, CDCl.sub.3) 7.60-7.06 (m, 20H), 5.18 (d, J=13.0 Hz, 2H), 4.99 (d, J=11.7 Hz, 1H), 4.79-4.69 (m, 3H), 4.64 (d, J=11.8 Hz, 1H), 4.50 (d, J=6.9 Hz, 2H), 3.98-3.85 (m, 2H), 3.83-3.73 (m, 1H), 3.62 (s, 1H), 3.58-3.45 (m, 1H), 3.37-3.15 (m, 3H), 1.58-1.41 (m, 4H), 1.31-1.16 (m, 5H); .sup.13C-NMR (100 MHz, CDCl.sub.3) 138.4, 137.8, 128.8, 128.7, 128.6, 128.3, 128.1, 127.9 (2C), 127.7, 127.4, 98.6, 77.2, 75.2, 74.9, 71.2, 67.7, 67.3, 66.9, 58.1, 50.6, 50.3, 47.2, 46.2, 29.1, 23.5, 16.9. 34*: [].sub.D.sup.20=+59.2 (c=0.9, CHCl.sub.3); IR v.sub.max (film) 2928, 2110, 1697, 1496, 1454, 1421, 1359, 1229, 1119, 1070 cm.sup.1; .sup.1H-NMR (400 MHz, CDCl.sub.3) 7.55-7.09 (m, 20H), 5.17 (d, J=12.4 Hz, 2H), 5.02 (d, J=11.9 Hz, 1H), 4.77-4.70 (m, 2H), 4.62 (d, J=12.2 Hz, 1H), 4.53-4.44 (m, 2H), 4.31 (d, J=14.7 Hz, 1H), 3.96-3.78 (m, 2H), 3.52 (s, 2H), 3.44-3.14 (m, 4H), 1.62-1.45 (m, 4H), 1.38-1.20 (m, 5H); .sup.13C-NMR (100 MHz, CDCl.sub.3) 138.6, 138.1, 137.6, 128.7, 128.6, 128.2, 128.1, 128.0, 127.9, 127.6 (2C), 127.4, 100.1, 78.8, 77.5, 77.2, 76.8, 74.8, 74.1, 71.8, 70.8, 69.3, 67.2, 58.8, 29.3, 23.4, 16.9; HRMS (MALDI-TOF): Calcd for C.sub.40H.sub.46N.sub.4O.sub.6Na.sup.+ [M+Na].sup.+ 701.3310, found 701.3337.

Example 35: Synthesis of N-(benzyl)benzyloxycarbonyl-5-amino-pentanyl 2-N-acetyl-3,4-di-O-benzyl-L-pneumosaminopyranosides (35* and 35*)

(156) ##STR00098##

(157) According to general procedure (F), azido-pneumosides 34* (28 mg, 41 mol) and 34* (10 mg, 15 mol) were individually reacted with thioacetic acid for 12 h to give 35* (19 mg, 27 mol, 66%) and 35* (7 mg, 10 mol, 68%). 35*: [].sub.D.sup.20=47.4 (c=1.9, CHCl.sub.3); IR v.sub.max (film) 3404, 2935, 1698, 1675, 1515, 1497, 1454, 1421, 1361, 1305, 1228, 1120, 1055, 1040 cm.sup.1; .sup.1H-NMR (400 MHz, CDCl.sub.3) 7.43-7.12 (m, 20H), 5.18 (d, J=14.2 Hz, 2H), 4.90 (d, J=10.3 Hz, 1H), 4.75-4.68 (m, 2H), 4.60 (d, J=10.3 Hz, 1H), 4.56-4.40 (m, 4H), 3.92-3.79 (m, 2H), 3.67-3.60 (m, 1H), 3.59-3.15 (m, 5H), 1.74 (s, 3H), 1.61-1.40 (m, 4H), 1.35-1.15 (m, 5H); .sup.13C-NMR (100 MHz, CDCl.sub.3) 170.4, 138.3, 138.2, 138.0, 128.7, 128.6, 128.5 (2C), 128.2, 128.1, 127.9, 127.7 (2C), 127.4, 127.3, 100.1, 78.7, 75.7, 72.8, 69.9, 67.7, 67.3, 66.3, 50.6, 50.3, 48.0, 47.2, 46.2, 29.3, 28.1, 27.7, 23.5, 16.8. 35*: [].sub.D.sup.20=+18.3 (c=0.7, CHCl.sub.3); IR v.sub.max (film) 3420, 2929, 2865, 1698, 1677, 1521, 1454, 1421, 1367, 1312, 1230, 1113, 1058 cm.sup.1; .sup.1H-NMR (400 MHz, CDCl.sub.3) 7.42-7.13 (m, 20H), 6.79 (d, J=9.8 Hz, 1H), 5.16 (d, J=11.6 Hz, 2H), 4.91 (d, J=10.2 Hz, 1H), 4.84-4.73 (m, 2H), 4.58 (d, J=10.2 Hz, 1H), 4.52-4.42 (m, 3H), 4.28 (d, J=13.1 Hz, 1H), 3.80-3.65 (m, 1H), 3.59-3.51 (m, 2H), 3.50-3.35 (m, 2H), 3.27-3.10 (m, 2H), 1.76 (s, 3H), 1.59-1.43 (m, 4H), 1.34-1.17 (m, 5H); .sup.13C-NMR (100 MHz, CDCl.sub.3) 170.7, 138.2, 138.1, 128.8, 128.6 (2C), 128.5, 128.1, 128.0, 127.9 (2C), 127.8, 127.3, 100.5, 77.7, 76.2, 75.7, 71.4, 69.8, 67.2, 47.9, 29.3, 23.6, 23.3, 16.8; HRMS (MALDI-TOF): Calcd for C.sub.42H.sub.50N.sub.2O.sub.7Na.sup.+ [M+Na].sup.+ 717.3510, found 717.3543.

Example 36: Synthesis of 5-amino-pentanyl 2-N-acetyl-L-pneumosaminopyranosides (36* and 37*)

(158) ##STR00099##

(159) According to general procedure (I), pneumosaminosides 35* (19 mg, 27 mol) and 35* (7 mg, 10 mol) were individually submitted to hydrogenolysis to give 36* (7 mg, 24 mol, 88%) and 37* (2 mg, 7 mol, 68%), respectively. 36*: .sup.1H-NMR (400 MHz, D.sub.2O) 4.75 (s, 1H), 4.13-3.93 (m, 3H), 3.78 (s, 1H), 3.71-3.61 (m, 1H), 3.55-3.44 (m, 1H), 3.03-2.91 (m, 2H), 2.01 (s, 3H), 1.74-1.57 (m, 4H), 1.49-1.35 (m, 2H), 1.22 (d, J=6.6 Hz, 3H); .sup.13C-NMR (150 MHz, D.sub.2O) 176.8, 101.6, 73.5, 70.2, 69.5, 66.6, 53.9, 42.0, 30.5, 29.2, 25.0, 18.1. 37*: .sup.1H-NMR (400 MHz, D.sub.2O) 4.61 (d, J=1.7 Hz, 1H), 4.29 (d, J=5.0 Hz, 1H), 3.88 (dd, J=4.5, 3.4 Hz, 1H), 3.81 (dt, J=9.9, 6.3 Hz, 1H), 3.71-3.58 (m, 3H), 3.00-2.93 (m, 2H), 2.02 (s, 3H), 1.68-1.54 (m, 4H), 1.42-1.33 (m, 2H), 1.25 (d, J=6.5 Hz, 3H); .sup.13C-NMR (150 MHz, D.sub.2O) 177.2, 102.4, 74.2, 72.9, 72.0, 70.1, 54.5, 42.0, 30.8, 29.3, 25.9, 25.2, 24.8, 18.0; HRMS (ESI): Calcd for C.sub.13H.sub.26N.sub.2O.sub.5Na.sup.+ [M+Na].sup.+ 313.1739, found 313.1750.

Example 37: Synthesis of thexyldimethylsilyl 3-O-acetyl-2-azido-2-deoxy--L-fucopyranoside (38*) and thexyldimethylsilyl 4-O-acetyl-2-azido-2-deoxy--L-fucopyranoside (39*)

(160) ##STR00100##

(161) To a solution of 2-azido-2-deoxy--L-fucopyranoside (200 mg, 0.60 mmol) in DCM/pyridine (4:1 (v/v), 10 mL), a solution of AcCl (51 L, 0.72 mmol) in DCM (1 mL) was added drop-wise at 0 C. and stirred for 6 h at the same temperature. The mixture was diluted with DCM and washed with sat. aq. NaHCO.sub.3. The combined organic layers were dried over MgSO.sub.4, filtered and concentrated. Column chromatography on silica gel (DCM/MeOH/acetone) afforded 38* (163 mg, 0.44 mmol, 72%). [].sub.D.sup.20=+4.4 (c=1.0, CHCl.sub.3); IR v.sub.max (film) 3483, 2958, 2868, 2111, 1747, 1726, 1465, 1370, 1251, 1171, 1145, 1069, 1039 cm.sup.1; .sup.1H-NMR (400 MHz, CDCl.sub.3) 4.66 (dd, J=10.8, 3.1 Hz, 1H), 4.50 (d, J=7.6 Hz, 1H), 3.77 (d, J=2.6 Hz, 1H), 3.61 (qd, J=6.4, 0.8 Hz, 1H), 3.55 (dd, J=10.8, 7.6 Hz, 1H), 2.16 (s, 3H), 1.66 (dt, J=13.7, 6.9 Hz, 1H), 1.28 (d, J=6.5 Hz, 3H), 0.89 (d, J=1.0 Hz, 3H), 0.88 (s, 9H), 0.19 (s, 6H); .sup.13C-NMR (100 MHz, CDCl.sub.3) 170.3, 97.3, 74.0, 70.4, 69.5, 63.5, 34.0, 25.0, 21.2, 20.1, 20.0, 18.6, 18.5, 16.4, 1.9, 3.2; HRMS (MALDI-TOF): Calcd for C.sub.16H.sub.31N.sub.3O.sub.5SiNa.sup.+ [M+Na].sup.+ 396.1925, found 396.1911. Thexyldimethylsilyl 4-O-acetyl-2-azido-2-deoxy--l-fucopyranoside (39*) (22 mg, 0.06 mmol, 10%) was obtained as a byproduct. [].sub.D.sup.20=+5.3 (c=1.3, CHCl.sub.3); IR v.sub.max (film) 3462, 2959, 2869, 2112, 1744, 1465, 1443, 1380, 1252, 1185, 1114, 1073 cm.sup.1; .sup.1H-NMR (400 MHz, CDCl.sub.3) 5.09 (dd, J=3.5, 1.1 Hz, 1H), 4.48 (d, J=7.6 Hz, 1H), 3.65 (qd, J=6.4, 1.2 Hz, 1H), 3.57 (dd, J=10.4, 3.6 Hz, 1H), 3.40 (dd, J=10.4, 7.6 Hz, 1H), 2.19 (s, 3H), 1.68 (dt, J=13.7, 6.9 Hz, 1H), 1.17 (d, J=6.5 Hz, 3H), 0.93-0.86 (m, 12H), 0.19 (d, J=1.6 Hz, 6H); .sup.13C-NMR (100 MHz, CDCl.sub.3) 171.7, 97.2, 71.9, 71.1, 69.4, 66.5, 34.0, 25.0, 21.0, 20.1, 20.0, 18.6, 18.3, 16.5, 1.9, 3.0; HRMS (MALDI-TOF): Calcd for C.sub.16H.sub.31N.sub.3O.sub.5SiNa.sup.+ [M+Na].sup.+ 396.1925, found 396.1954.

Example 38: Synthesis of thexyldimethylsilyl 2-O-benzoyl-3,4,6-tri-O-benzyl--D-glucopyranosyl-(13)-4-O-acetyl-2-azido-2-deoxy--L-fucopyranoside 40*)

(162) ##STR00101##

(163) According to general procedure (B), 2-O-benzoyl-3,4,6-tri-O-benzyl-1-thio--D-glucopyranoside (109 mg, 0.18 mmol) was reacted with fucoside 39* (62 mg, 0.17 mmol), NIS (45 mg, 0.20 mmol) and TfOH (1.8 L, 20 mol) in DCM (1 mL) at 25 C. to 20 C. over 1 h. After workup column chromatography (hexanes/EtOAc) afforded 40* (139 mg, 0.15 mmol, 92%). [].sub.D.sup.20=13.9 (c=1.9, CHCl.sub.3); IR v.sub.max (film) 2958, 2867, 2113, 1744, 1496, 1453, 1364, 1267, 1234, 1179, 1095, 1071 cm.sup.1; .sup.1H-NMR (400 MHz, CDCl.sub.3) 8.10-8.03 (m, 2H), 7.60-7.54 (m, 1H), 7.50-7.42 (m, 2H), 7.41-7.21 (m, 10H), 7.16-7.02 (m, 5H), 5.32 (dd, J=9.1, 7.7 Hz, 1H), 5.12 (d, J=2.8 Hz, 1H), 4.82 (d, J=11.0 Hz, 1H), 4.76-4.66 (m, 4H), 4.63 (dd, J=11.0, 6.5 Hz, 2H), 4.36 (d, J=7.7 Hz, 1H), 3.89 (dd, J=10.4, 3.5 Hz, 1H), 3.86-3.80 (m, 2H), 3.79-3.72 (m, 2H), 3.62 (ddd, J=9.4, 5.3, 1.9 Hz, 1H), 3.50-3.41 (m, 2H), 1.73-1.62 (m, 1H), 1.49 (s, 3H), 1.07 (d, J=6.4 Hz, 3H), 0.92-0.87 (m, 12H), 0.17 (d, J=4.6 Hz, 6H); .sup.13C-NMR (100 MHz, CDCl.sub.3) 170.6, 164.9, 138.6, 138.0, 137.8, 133.3, 130.2, 129.8, 128.6, 128.5, 128.4, 128.3, 128.2, 128.1, 128.0, 127.7, 127.6 (2C), 97.1, 96.8, 83.0, 78.1, 75.7, 75.3, 75.1, 74.6, 73.7, 73.5, 69.0, 68.9, 67.8, 63.9, 34.0, 25.0, 20.2, 20.1 (2C), 18.6 (2C), 16.5, 1.8, 3.0; HRMS (MALDI-TOF): Calcd for C.sub.50H.sub.63N.sub.3O.sub.11SiNa.sup.+ [M+Na].sup.+ 932.4124, found 932.4125.

Example 39: Synthesis of 2-O-benzoyl-3,4,6-tri-O-benzyl--d-glucopyranosyl-(13)-4-O-acetyl-2-azido-2-deoxy--l-fucopyranosyl trichloroacetimidate (41*)

(164) ##STR00102##

(165) According to general procedure (A), disaccharide 40* (55 mg, 60 mol) was reacted with TBAF (1 m in THF, 600 L, 600 mol) and AcOH (42 L, 725 mol) in THF (2 mL) to give the crude lactol. According to general procedure (D), the crude lactol was reacted with trichloroacetonitrile (60 L, 600 mol) and DBU in DCM (2 mL) at 0 C. to afford 41* (44 mg, 48 mmol, 81%). [].sub.D.sup.20=64.8 (c=1.8, CHCl.sub.3)IR v.sub.max (film) 2871, 2115, 1743, 1673, 1496, 1453, 1361, 1267, 1229, 1093, 1070, 1027 cm.sup.1; .sup.1H-NMR (400 MHz, CDCl.sub.3) 8.67 (s, 1H), 8.11-8.03 (m, 2H), 7.60-7.56 (m, 1H), 7.50-7.43 (m, 2H), 7.42-7.26 (m, 8H), 7.24-7.18 (m, 2H), 7.16-7.03 (m, 5H), 6.35 (d, J=3.6 Hz, 1H), 5.38-5.30 (m, 2H), 4.84-4.78 (m, 2H), 4.73 (d, J=11.0 Hz, 1H), 4.67-4.54 (m, 5H), 4.21 (q, J=6.4 Hz, 1H), 3.88-3.71 (m, 5H), 3.61 (ddd, J=9.5, 4.7, 1.8 Hz, 1H), 1.44 (s, 3H), 1.08 (d, J=6.5 Hz, 3H); .sup.13C-NMR (100 MHz, CDCl.sub.3) 170.3, 165.0, 160.9, 138.3, 137.9, 137.8, 133.4, 130.3, 129.7, 128.6, 128.5 (2C), 128.4, 128.2, 128.1, 128.0, 127.8, 127.7, 96.8, 95.4, 91.1, 82.9, 78.1, 75.8, 75.4, 75.2, 73.9, 73.7, 70.8, 69.0, 68.2, 68.0, 57.2, 19.9, 16.3; HRMS (ESI): Calcd for C.sub.44H.sub.45Cl.sub.3N.sub.4O.sub.11Na.sup.+ [M+Na].sup.+ 933.2048, found 933.2051.

Example 40: Synthesis of N-(benzyl)benzyloxycarbonyl-5-amino-pentanyl 2-O-benzoyl-3,4,6-tri-O-benzyl--D-glucopyranosyl-(13)-4-O-acetyl-2-azido-2-deoxy-L-fucopyranoside (42*)

(166) ##STR00103##

(167) According to general procedure (E), disaccharide-imidate 41* (44 mg, 48 mol) and N-(benzyl)benzyloxycarbonyl-5-amino-pentanol (32 mg, 96 mol) were reacted in DCM (2 mL) at 30 C. to 20 C. over 30 min to give the and anomers of 42* (45 mg, 42 mol, 87%) in a ratio /=1:6. Analytical data is given for the anomer. [].sub.D.sup.20=68.5 (c=0.5, CHCl.sub.3); IR v.sub.max (film) 2927, 2111, 1743, 1698, 1454, 1267, 1091 cm.sup.1; .sup.1H-NMR (400 MHz, CDCl.sub.3) 8.06 (d, J=7.9 Hz, 2H), 7.57 (t, J=7.4 Hz, 1H), 7.44 (d, J=7.6 Hz, 2H), 7.39-7.27 (m, 15H), 7.23-7.01 (m, 10H), 5.31 (t, J=8.4 Hz, 1H), 5.25 (s, 1H), 5.16 (d, J=7.8 Hz, 2H), 4.85-4.78 (m, 2H), 4.74-4.69 (m, 2H), 4.63-4.52 (m, 4H), 4.45 (s, 3H), 4.01-3.90 (m, 1H), 3.85-3.69 (m, 4H), 3.59 (dd, J=8.6, 4.1 Hz, 2H), 3.41 (dd, J=10.8, 3.6 Hz, 2H), 3.18 (d, J=20.7 Hz, 2H), 1.56-1.43 (m, 4H), 1.42 (s, 3H), 1.35-1.18 (m, 3H), 1.02 (d, J=6.4 Hz, 3H); .sup.13C-NMR (100 MHz, CDCl.sub.3) 170.5, 165.0, 138.4, 138.0, 137.8, 133.3, 130.3, 129.8, 128.7, 128.6, 128.5, 128.4, 128.3, 128.2, 128.1, 128.0, 127.9, 127.7 (2C), 98.6, 97.4, 83.1, 78.1, 77.4, 75.7, 75.4, 75.2, 73.7, 73.6, 70.9, 69.1, 68.9, 68.5, 67.3, 65.0, 57.8, 50.6, 47.2, 46.2, 29.2, 23.4, 20.0, 16.3; HRMS (ESI): Calcd for C.sub.62H.sub.68N.sub.4O.sub.13Na.sup.+ [M+Na].sup.+ 1099.4681, found 1099.4679.

Example 41: Synthesis of N-(benzyl)benzyloxycarbonyl-5-amino-pentanyl 2-O-benzoyl-3,4,6-tri-O-benzyl--D-glucopyranosyl-(13)-2-N-acetyl-4-O-acetyl--L-fucosaminopyranoside (43*)

(168) ##STR00104##

(169) According to general procedure (F), azido-disaccharide 42* (10 mg, 9 mol) was reacted with thioacetic acid for 48 h to give 43* (8 mg, 7 mol, 79%). [].sub.D.sup.20=5.5 (c=1.0, CHCl.sub.3); IR v.sub.max (film) 2928, 1743, 1697, 1454, 1365, 1266, 1232, 1091 cm.sup.1; .sup.1H-NMR (400 MHz, CDCl.sub.3) 8.06-8.01 (m, 2H), 7.61-7.55 (m, 1H), 7.47-7.42 (m, 2H), 7.38-7.26 (m, 15H), 7.26-7.20 (m, 2H), 7.18-7.06 (m, 8H), 6.50-6.27 (m, 1H), 5.22-5.12 (m, 5H), 4.80 (d, J=10.9 Hz, 1H), 4.69 (d, J=10.9 Hz, 1H), 4.66-4.60 (m, 2H), 4.50 (dd, J=22.3, 11.3 Hz, 5H), 4.08 (s, 2H), 3.98-3.86 (m, 1H), 3.82-3.49 (m, 6H), 3.43-3.28 (m, 1H), 3.28-3.06 (m, 2H), 1.83 (s, 3H), 1.64 (s, 3H), 1.55-1.42 (m, 4H), 1.29-1.16 (m, 2H), 1.01 (d, J=6.5 Hz, 3H); .sup.13C-NMR (100 MHz, CDCl.sub.3) 170.7, 165.1, 138.0, 137.8, 137.6, 133.3, 130.1, 129.8, 128.7 (2C), 128.6, 128.4 (2C), 128. (2C), 127.9, 127.8, 98.9, 97.1, 82.9, 77.8, 77.4, 75.2 (2C), 74.5, 73.5, 73.3, 72.6, 69.9, 68.9, 68.3, 67.3, 64.8, 49.7, 29.2, 23.4, 23.1, 20.2, 16.3; HRMS (MALDI-TOF): Calcd for C.sub.64H.sub.72N.sub.2O.sub.14Na.sup.+ [M+Na].sup.+ 1115.4876, found 1115.4890.

Example 42: Synthesis of 5-amino-pentanyl -D-glucopyranosyl-(13)-2-N-acetyl--L-fucosaminopyranoside (44*)

(170) ##STR00105##

(171) To a solution of 43* (8 mg, 7 mol) in MeOH (1 mL) was added 0.5 m NaOMe in MeOH (0.2 mL) and stirred for 16 h. The mixture was neutralized with Amberlite IR 120 (H.sup.+) ion exchange resin and the resulting diol was filtered and concentrated. According to general procedure (I), the crude diol was submitted to hydrogenolysis to give 44* (3.4 mg, 7.5 mol, 89%). .sup.1H-NMR (600 MHz, D.sub.2O) 4.95 (d, J=3.7 Hz, 1H), 4.55 (d, J=7.9 Hz, 1H), 4.19 (dd, J=11.3, 3.7 Hz, 1H), 4.13 (dd, J=11.3, 2.9 Hz, 1H), 4.09 (q, J=6.6 Hz, 1H), 4.03 (d, J=2.5 Hz, 1H), 3.98 (dd, J=12.2, 2.1 Hz, 1H), 3.75-3.68 (m, 2H), 3.54-3.45 (m, 3H), 3.38 (t, J=9.5 Hz, 1H), 3.31 (dd, J=9.4, 8.0 Hz, 1H), 3.03-2.98 (m, 2H), 2.05 (s, 3H), 1.73-1.62 (m, 4H), 1.49-1.41 (m, 2H), 1.27 (d, J=6.6 Hz, 3H); .sup.13C-NMR (150 MHz, D.sub.2O) 177.1, 103.0, 99.3, 78.7, 78.1, 77.8, 75.4, 72.3, 71.3, 70.4, 69.0, 63.6, 50.9, 42.0, 30.5, 29.0, 24.8, 24.6, 18.1; HRMS (ESI): Calcd for C.sub.19H.sub.36N.sub.2O.sub.10Na.sup.+ [M+Na].sup.+ 475.2268, found 475.2273.

Example 43: Synthesis of thexyldimethylsilyl 3,4-di-O-benzyl-2-O-levulinoyl-6-O-(2-naphthalenylmethyl)--d-glucopyranosyl-(14)-3-O-acetyl-2-azido-2-deoxy--L-fucopyranoside (45*)

(172) ##STR00106##

(173) According to general procedure (B), thioglucoside 10* (140 mg, 218 mol) was reacted with fucoside 38* (50 mg, 134 mol), NIS (54 mg, 240 mol) and TfOH (2.4 L, 27 mol) in DCM (1 mL) at 30 C. to 20 C. over 1 h. After work-up, column chromatography (hexanes/EtOAc) afforded 45* (114 mg, 119 mol, 89%). [].sub.D.sup.20=13.0 (c=1.8, CHCl.sub.3); IR v.sub.max (film) 2958, 2867, 2112, 1748, 1721, 1603, 1509, 1455, 1364, 1240, 1147, 1072 cm.sup.1; .sup.1H-NMR (400 MHz, CDCl.sub.3) 7.87-7.76 (m, 4H), 7.52-7.43 (m, 3H), 7.35-7.25 (m, 5H), 7.23-7.12 (m, 3H), 7.04-6.98 (m, 2H), 5.16 (dd, J=9.6, 8.1 Hz, 1H), 4.80-4.75 (m, 3H), 4.70 (d, J=12.1 Hz, 1H), 4.60 (d, J=12.2 Hz, 1H), 4.49-4.39 (m, 3H), 4.30 (d, J=8.1 Hz, 1H), 3.94 (d, J=3.4 Hz, 1H), 3.85-3.79 (m, 1H), 3.76-3.65 (m, 3H), 3.58 (dd, J=11.0, 7.6 Hz, 1H), 3.52 (q, J=6.5 Hz, 1H), 3.39 (ddd, J=9.8, 3.5, 2.0 Hz, 1H), 2.93-2.81 (m, 1H), 2.68-2.63 (m, 1H), 2.62-2.58 (m, 1H), 2.54-2.45 (m, 1H), 2.16 (s, 3H), 2.11 (s, 3H), 1.67 (dt, J=13.7, 6.9 Hz, 1H), 1.25 (d, J=6.5 Hz, 3H), 0.92 (s, 3H), 0.90 (s, 3H), 0.89 (s, 6H), 0.18 (s, 3H), 0.15 (s, 3H); .sup.13C-NMR (100 MHz, CDCl.sub.3) 206.4, 171.4, 171.2, 138.3, 137.9, 135.1, 133.3, 133.1, 128.4 (3C), 128.1, 128.0, 127.9, 127.8 (3C), 126.6, 126.3, 126.1, 125.8, 101.9, 97.2, 82.9, 77.5, 75.1, 75.0 (2C), 74.3, 73.8, 73.4, 72.8, 70.0, 68.7, 62.8, 38.1, 34.0, 29.9, 28.0, 24.9, 21.0, 20.0 (2C), 18.6, 18.5, 16.4, 2.0, 3.1; HRMS (MALDI-TOF): Calcd for C.sub.52H.sub.67N.sub.3O.sub.12SiNa.sup.+ [M+Na].sup.+ 976.4386, found 976.4333.

Example 44: Synthesis of 3,4-di-O-benzyl-2-O-levulinoyl-6-O-(2-naphthalenylmethyl)--D-glucopyranosyl-(14)-3-O-acetyl-2-azido-2-deoxy-L-fucopyranoside (46*)

(174) ##STR00107##

(175) According to general procedure (A), disaccharide 45* (105 mg, 0.11 mmol) was reacted with TBAF (1.0 mL, 1.00 mmol) and AcOH (70 L, 1.22 mmol) in THF (2 mL) to give lactol 46* (81 mg, 0.10 mmol, 95%) as a mixture of and anomers. IR v.sub.max(film) 3424, 2869, 2111, 1742, 1717, 1454, 1363, 1237, 1151, 1059 cm.sup.1; .sup.1H-NMR (400 MHz, CDCl.sub.3) 7.87-7.75 (m, 4H), 7.53-7.41 (m, 3H), 7.34-7.25 (m, 5H), 7.22-7.10 (m, 3H), 7.03-6.95 (m, 2H), 5.37-5.28 (m, 0.5H) 5.25 (d, J=3.5 Hz, 0.5H), 5.16-5.02 (m, 1.5H), 4.80-4.65 (m, 4H), 4.60 (d, J=12.1 Hz, 1H), 4.50-4.43 (m, 2H), 4.35 (d, J=8.0 Hz, 0.5H), 4.31 (d, J=8.1 Hz, 0.5H), 4.19 (q, J=6.6 Hz, 0.5H), 4.07 (dd, J=11.1, 3.1 Hz, 1H), 3.88-3.56 (m, 6H), 3.41-3.35 (m, 1H), 2.92-2.62 (m, 2H), 2.55-2.44 (m, 1H), 2.24 (s, 1.5H), 2.15 (s, 1.5H), 2.12 (s, 1.5H), 2.09 (s, 1.5H), 1.29 (d, J=6.5 Hz, 1.5H), 1.19 (d, J=6.6 Hz, 1.5H); .sup.13C-NMR (100 MHz, CDCl.sub.3) 208.9, 206.4, 171.9, 171.7, 171.2, 171.1, 138.5, 138.4, 137.9, 135.1 (2C), 133.3, 133.1, 128.5 (2C), 128.4 (2C), 128.1, 127.9 (4C), 127.8 (3C), 127.7, 126.8, 126.6, 126.4, 126.1, 125.9, 125.8, 101.7, 100.9, 96.8, 92.2, 83.0, 82.9, 77.8, 77.6, 75.4, 75.2, 75.1, 75.0 (2C), 73.9 (2C), 73.6, 73.3, 72.8, 70.7, 70.4, 68.8, 68.6, 65.8, 62.8, 57.9, 38.1, 37.6, 30.5, 30.0, 28.1 (2C), 21.1, 21.0, 17.0, 16.3; HRMS (MALDI-TOF): Calcd for C.sub.44H.sub.49N.sub.3O.sub.12Na.sup.+ [M+Na].sup.+ 834.3208, found 834.3222.

Example 45: Synthesis of 3,4-di-O-benzyl-2-O-levulinoyl-6-O-(2-naphthalenylmethyl)--D-glucopyranosyl-(14)-3-O-acetyl-2-azido-2-deoxy-L-fucopyranosyl trichloroacetimidate (47*)

(176) ##STR00108##

(177) According to general procedure (D), lactol 46* (81 mg, 100 mol) was reacted with trichloroacetonitrile (100 L, 998 mol) and DBU in DCM (2 mL) at 0 C. to afford 47* (82 mg, 86 mmol, 86%) as a mixture of and anomers. Analytical data is given for the anomer. [].sub.D.sup.20=88.7 (c=1.5, CHCl.sub.3); IR v.sub.max (film) 3336, 3060, 3030, 2906, 2868, 2113, 1746, 1718, 1673, 1363, 1273, 1243, 1144, 1063, 1028 cm.sup.1; .sup.1H-NMR (400 MHz, CDCl.sub.3) 8.67 (s, 1H), 7.90-7.75 (m, 4H), 7.52-7.42 (m, 3H), 7.35-7.25 (m, 5H), 7.23-7.10 (m, 3H), 7.05-6.96 (m, 2H), 6.36 (d, J=3.6 Hz, 1H), 5.14 (dd, J=9.6, 8.1 Hz, 1H), 5.08 (dd, J=11.1, 3.1 Hz, 1H), 4.80-4.69 (m, 4H), 4.60 (d, J=12.1 Hz, 1H), 4.47 (d, J=10.8 Hz, 1H), 4.34 (d, J=8.0 Hz, 1H), 4.22-4.13 (m, 2H), 4.10 (dd, J=11.1, 3.6 Hz, 1H), 3.87-3.72 (m, 3H), 3.71-3.64 (m, 1H), 3.40 (ddd, J=9.8, 3.5, 2.0 Hz, 1H), 2.84-2.64 (m, 2H), 2.61-2.46 (m, 2H), 2.17 (s, 3H), 2.13 (s, 3H), 1.24 (d, J=6.6 Hz, 3H); .sup.13C-NMR (100 MHz, CDCl.sub.3) 206.3, 171.7, 171.0, 161.1, 138.3, 137.9, 135.0, 133.3, 133.2, 128.5, 128.4, 128.1, 128.0, 127.9, 127.8 (2C), 126.7, 126.4, 126.1, 125.9, 101.7, 95.2, 91.1, 82.9, 77.5, 75.1, 74.5, 73.9, 73.5, 70.7, 68.7, 68.5, 56.6, 38.1, 30.0, 28.1, 21.0, 16.3; HRMS (ESI): Calcd for C.sub.46H.sub.49Cl.sub.3N.sub.4O.sub.12Na.sup.+ [M+Na].sup.+ 977.2310, found 977.2312.

Example 46: Synthesis of N-(benzyl)benzyloxycarbonyl-5-amino-pentanyl 3,4-di-O-benzyl-2-O-levulinoyl-6-O-(2-naphthalenylmethyl)--d-glucopyranosyl-(14)-3-O-acetyl-2-azido-2-deoxy-L-fucopyranoside (48*)

(178) ##STR00109##

(179) According to general procedure (E), disaccharide-imidate 47* (17 mg, 18 mol) and N-(benzyl)benzyloxycarbonyl-5-amino-pentanol (12 mg, 36 mol) were reacted in DCM (1 mL) at 30 C. to 20 C. over 30 min to give the and anomers of 48* (18 mg, 16 mol, 90%) in a ratio /=1:3. Analytical data is given for the anomer. [].sub.D.sup.20=70.3 (c=0.3, CHCl.sub.3); IR v.sub.max (film) 2936, 2110, 1746, 1699, 1497, 1454, 1421, 1362, 1244, 1131, 1054 cm.sup.1; .sup.1H-NMR (400 MHz, CDCl.sub.3) 7.84-7.73 (m, 4H), 7.49-7.40 (m, 3H), 7.34-7.21 (m, 14H), 7.19-7.09 (m, 4H), 7.00-6.95 (m, 2H), 5.18-5.07 (m, 3H), 5.00 (dd, J=11.1, 2.6 Hz, 1H), 4.78-4.65 (m, 5H), 4.57 (d, J=12.0 Hz, 1H), 4.51-4.42 (m, 3H), 4.28 (d, J=7.9 Hz, 1H), 4.04 (s, 1H), 3.92-3.86 (m, 1H), 3.77 (d, J=9.4 Hz, 1H), 3.72-3.57 (m, 4H), 3.36 (d, J=9.6 Hz, 2H), 3.26-3.15 (m, 2H), 2.82-2.57 (m, 3H), 2.50 (t, J=7.0 Hz, 2H), 2.13 (s, 3H), 2.08 (s, 3H), 1.54-1.46 (m, 4H), 1.33-1.25 (m, 2H), 1.15 (d, J=6.4 Hz, 3H); .sup.13C-NMR (100 MHz, CDCl.sub.3) 206.4, 171.7, 171.2, 138.4, 137.9, 135.1, 133.4, 133.2, 128.7, 128.5 (2C), 128.1, 128.0, 127.9 (3C), 127.8, 127.4, 126.7, 126.4, 126.1, 125.9, 101.7, 98.0, 83.0, 77.6, 77.4, 75.4, 75.1 (2C), 75.0, 73.9, 73.5, 70.3, 68.7, 68.3, 67.3, 65.6, 57.1, 38.2, 30.0, 29.3, 28.1, 23.5, 21.1, 16.3; HRMS (MALDI-TOF): Calcd for C.sub.64H.sub.72N.sub.4O.sub.14Na.sup.+ [M+Na].sup.+ 1143.4937, found 1143.4974.

Example 47: Synthesis of N-(benzyl)benzyloxycarbonyl-5-amino-pentanyl 3,4-di-O-benzyl-2-O-levulinoyl-6-O-(2-naphthalenylmethyl)--D-glucopyranosyl-(14)-2-N-acetyl-3-O-acetyl--L-fucosaminopyranoside (49*)

(180) ##STR00110##

(181) According to general procedure (F), azido-disaccharide 48* (28 mg, 25 mol) was reacted with thioacetic acid for 24 h to give 49* (20 mg, 18 mol, 70%). [].sub.D.sup.20=53.6 (c=1.0, CHCl.sub.3); IR v.sub.max (film) 2926, 1694, 1454, 1364, 1247, 1053 cm.sup.1; .sup.1H-NMR (400 MHz, CDCl.sub.3) 7.92-7.72 (m, 4H), 7.60-7.08 (m, 21H), 7.06-6.90 (m, 2H), 5.90-5.30 (m, 1H), 5.27-5.02 (m, 3H), 4.84 (d, J=11.4 Hz, 1H), 4.80-4.67 (m, 5H), 4.64-4.57 (m, 1H), 4.56-4.39 (m, 4H), 4.34 (d, J=8.0 Hz, 1H), 3.99-3.46 (m, 7H), 3.39 (d, J=9.7 Hz, 1H), 3.36-3.10 (m, 3H), 2.89-2.62 (m, 2H), 2.62-2.44 (m, 2H), 2.15 (s, 3H), 2.04 (s, 3H), 1.92 (s, 3H), 1.66-1.41 (m, 4H), 1.39-1.22 (m, 2H), 1.19 (d, J=5.6 Hz, 3H); .sup.13C-NMR (100 MHz, CDCl.sub.3) 206.8, 171.9, 171.3, 171.1, 138.4, 137.9, 135.3, 133.4, 133.1, 128.7, 128.6, 128.5 (2C), 128.4, 128.3, 128.1, 128.0 (2C), 127.8 (2C), 127.7, 126.5, 126.3, 126.0, 125.9, 101.8, 97.5, 83.2, 77.7, 77.4, 75.5, 75.1, 74.9, 73.9, 73.5, 70.3, 69.0, 67.3, 65.6, 50.3, 47.5, 47.2, 38.2, 38.0, 30.0, 29.3, 28.1, 27.5, 23.9, 23.5, 21.2, 16.4; HRMS (MALDI-TOF): Calcd for C.sub.66H.sub.76N.sub.2O.sub.15Na.sup.+ [M+Na].sup.+ 1159.5138, found 1159.5148.

Example 48: Synthesis of N-(benzyl)benzyloxycarbonyl-5-amino-pentanyl 3,4-di-O-benzyl--D-glucopyranosyl-(14)-2-N-acetyl--L-fucosaminopyranoside (50*)

(182) ##STR00111##

(183) According to general procedure (G), disaccharide 49* (20 mg, 18 mol) was reacted with DDQ (12 mg, 53 mol) for 1.5 h to give the crude primary alcohol. To a solution of crude primary alcohol in MeOH (1 mL) was added 0.5 m NaOMe in MeOH (0.3 mL) and stirred for 16 h. The mixture was neutralized with Amberlite IR 120 (H.sup.+) ion exchange resin, filtered and concentrated. Column chromatography (DCM/MeOH/acetone) afforded 50* (9 mg, 11 mol, 60%). [].sub.D.sup.20=45.2 (c=0.7, CHCl.sub.3); IR v.sub.max (film) 3354, 2925, 1695, 1542, 1497, 1454, 1422, 1361, 1231, 1049 cm.sup.1; .sup.1H-NMR (400 MHz, CDCl.sub.3) 7.68-6.84 (m, 20H), 6.07-5.61 (m, 1H), 5.18 (d, J=11.9 Hz, 2H), 5.01-4.76 (m, 3H), 4.76-4.58 (m, 2H), 4.57-4.33 (m, 4H), 4.02-3.80 (m, 2H), 3.80-3.42 (m, 8H), 3.39-3.14 (m, 3H), 2.53 (s, 1H), 2.01 (s, 3H), 1.91-1.63 (m, 2H), 1.63-1.45 (m, 4H), 1.36-1.26 (m, 5H); .sup.13C-NMR (100 MHz, CDCl.sub.3) 172.0, 138.6, 137.9, 128.7, 128.6 (2C), 128.3, 128.1, 128.0, 127.9 (2C), 103.4, 98.1, 84.3, 83.3, 77.7, 77.4, 76.3, 75.4, 75.2, 74.9, 70.5, 67.4, 67.2, 61.9, 50.3, 47.0, 46.2, 29.3, 27.5, 23.7, 16.8; HRMS (MALDI-TOF): Calcd for C.sub.48H.sub.60N.sub.2O.sub.12Na.sup.+ [M+Na].sup.+ 879.4038, found 879.4004.

Example 50: Synthesis of 5-amino-pentanyl -D-glucopyranosyluronate-(14)-2-N-acetyl--L-fucosaminopyranoside (51*)

(184) ##STR00112##

(185) According to general procedure (H), triol 50* (8 mg, 9 mol) was reacted with TEMPO (0.3 mg, 2 mol) and BAIB (15 mg, 47 mol) to give the protected uronate disaccharide. According to general procedure (I), the uronate disaccharide was subjected to hydrogenolysis to give carboxylic acid 51* (2.6 mg, 5.3 mol, 57%). .sup.1H-NMR (600 MHz, D.sub.2O) 4.89 (d, J=3.7 Hz, 1H), 4.53 (d, J=7.3 Hz, 1H), 4.20-4.12 (m, 2H), 4.05 (d, J=2.9 Hz, 1H), 3.93 (dd, J=11.2, 3.0 Hz, 1H), 3.77-3.72 (m, 1H), 3.70 (dt, J=8.1, 5.6 Hz, 1H), 3.59-3.45 (m, 4H), 3.06-2.97 (m, 2H), 2.05 (s, 3H), 1.73-1.63 (m, 4H), 1.49-1.42 (m, 2H), 1.34 (d, J=6.6 Hz, 3H); .sup.13C-NMR (150 MHz, D.sub.2O) 178.1, 177.1, 105.5, 99.6, 82.5, 78.8, 77.9, 75.8, 74.4, 70.5, 69.6, 69.5, 53.2, 42.0, 30.6, 29.1, 24.9, 24.5, 17.9; HRMS (ESI): Calcd for C.sub.19H.sub.34N.sub.2O.sub.11Na.sup.+ [M+Na].sup.+ 489.2060, found 489.2063.

Example 51: Synthesis of 5-amino-pentanyl 2-N-acetyl-L-fucosaminopyranoside (52*)

(186) ##STR00113##

(187) The fucosamine derivative 52* was obtained according to the synthetic procedure described for the synthesis of pneumosamine derivative 36* (3.26 mg, 11 mol, 83% yield).sup.1H-NMR (400 MHz, D.sub.2O) 4.81 (d, J=3.8 Hz, 1H), 4.11-4.02 (m, 2H), 3.88 (dd, J=11.1, 3.3 Hz, 1H), 3.77 (d, J=3.1 Hz, 1H), 3.65 (dt, J=10.1, 6.6 Hz, 1H), 3.44 (dt, J=10.1, 6.3 Hz, 1H), 3.01-2.92 (m, 2H), 2.01 (s, 3H), 1.70-1.57 (m, 4H), 1.47-1.35 (m, 2H), 1.20 (d, J=6.6 Hz, 3H); .sup.13C-NMR (100 MHz, D.sub.2O) 174.4, 96.8, 71.0, 67.7, 67.7, 66.5, 49.6, 39.2, 27.9, 26.4, 22.2, 21.8, 15.3; HRMS (ESI): Calcd for C.sub.13H.sub.26N.sub.2O.sub.5Na.sup.+ [M+Na].sup.+ 313.1739, found 313.1750.

Example 52: Synthesis of thexyldimethylsilyl 3,4,6-tri-O-benzyl-2-O-levulinoyl--D-glucopyranosyl-(14)-3-O-acetyl-2-azido-2-deoxy--L-fucopyranoside (53*)

(188) ##STR00114##

(189) Alcohol 38* (100 mg, 0.268 mmol, 1.0 equiv) and 2-O-levulinoyl-3,4,6-tri-O-benzyl-1-thio--D-glucopyranoside (190 mg, 0.321 mmol, 1.2 equiv) were co-evaporated with toluene two times and dried in vacuo. The residue was dissolved in dry dichloromethane. Molecular sieves (4 ) were added and the reaction mixture cooled to 30 C. NIS (73 mg, 0.321 mmol, 1.2 equiv) and TfOH (3 L, 0.032 mmol, 0.12 equiv) were added and the reaction let warm to 20 C. over one hour. The reaction was quenched with triethylamine and diluted with dichloromethane. The organic phase was washed with saturated aqueous Na.sub.2S.sub.2O.sub.3, NaHCO.sub.3 and brine. The organic layer was dried over Na.sub.2SO.sub.4 and concentrated. Column chromatography on silica gel (hexanes/ethyl acetate) afforded disaccharide 53* (182 mg, 0.201 mmol, 75%). .sup.1H NMR (400 MHz, CDCl.sub.3) 7.40-7.25 (m, 13H), 7.15-7.12 (m, 2H), 5.14 (dd, J=9.5, 8.2 Hz, 1H), 4.79 (d, J=10.8 Hz, 1H), 4.76 (s, 2H), 4.59-4.35 (m, 5H), 4.30 (d, J=8.1 Hz, 1H), 3.93 (d, J=3.3 Hz, 1H), 3.81 (t, J=9.3 Hz, 1H), 3.75-3.64 (m, 3H), 3.57 (dd, J=10.9, 7.7 Hz, 1H), 3.52 (dd, J=12.9, 6.4 Hz, 1H), 3.40-3.34 (m, 1H), 2.93-2.42 (m, 4H), 2.16 (s, 3H), 2.09 (s, 3H), 1.73-1.59 (m, 1H), 1.26 (d, J=6.5 Hz, 3H), 0.93-0.85 (m, 12H), 0.17 (s, 3H), 0.15 (m, 3H).

Example 53: Synthesis of thexyldimethylsilyl 3,4,6-tri-O-benzyl--D-glucopyranosyl-(14)-3-O-acetyl-2-azido-2-deoxy--L-fucopyranoside (54*)

(190) ##STR00115##

(191) To a solution of disaccharide 53* (180 mg, 0.199 mmol, 1 equiv) in DCM (11 mL) hydrazine hydrate (40 L, 0.796 mmol, 4 equiv) dissolved in AcOH (0.22 mL) and pyridine (0.33 mL) was added and the solution stirred for 1 h. The reaction was then quenched by the addition of acetone and concentrated. Column chromatography on silica gel (hexanes/ethyl acetate) afforded disaccharide 54* (150 mg, 0.186 mmol, 93%). .sup.1H NMR (400 MHz, CDCl.sub.3) 7.38-7.21 (m, 13H), 7.15-7.10 (m, 2H), 5.02 (d, J=11.2 Hz, 1H), 4.83 (d, J=11.0 Hz, 1H), 4.81 (d, J=11.4 Hz, 1H), 4.57-4.32 (m, 5H), 4.20 (d, J=7.7 Hz, 1H), 4.00 (d, J=3.3 Hz, 1H), 3.80-3.50 (m, 7H), 3.44-3.30 (m, 1H), 3.21 (s, 1H), 2.11 (s, 3H), 1.74-1.63 (m, 1H), 1.33 (d, J=6.5 Hz, 3H), 0.94-0.85 (m, 12H), 0.2 (s, 6H).

Example 54: Synthesis of thexyldimethylsilyl 3,4,6-tri-O-benzyl--D-glucopyranosyl-(14)-3-O-acetyl-2-azido-2-deoxy--L-fucopyranoside (55*)

(192) ##STR00116##

(193) Benzyl bromide (0.074 mL, 0.620 mmol, 10 equiv) was added to a solution of alcohol 55* (50 mg, 0.062 mmol, 1 equiv) in DCM (0.5 mL). Then, the mixture was cooled to 0 C. and NaH 60% in mineral oil (304 mg, 7.60 mmol) was added. After 10 min, DMF was added (17 L). The reaction was warmed to room temperature and let stir for 30 min. The reaction mixture was cooled to 0 C. and quenched with AcOH (0.1 mL). The reaction mixture was warmed to room temperature and the solvent evaporated. Column chromatography on silica gel (hexanes/ethyl acetate) afforded disaccharide 55* (55 mg, 0.061 mmol, 99%). .sup.1H NMR (400 MHz, CDCl.sub.3) 7.45-7.03 (m, 20H), 5.19 (dd, J=9.6, 8.1 Hz, 1H), 4.89 (d, J=12.6 Hz, 1H), 4.82-4.75 (m, 2H), 4.70 (d, J=11.4 Hz, 1H), 4.56-4.44 (m, 3H), 4.40 (d, J=12.0 Hz, 1H), 4.35 (d, J=12.0 Hz, 1H), 4.31 (d, J=7.7 Hz, 1H), 3.87 (d, J=3.0 Hz, 1H), 3.73-3.43 (m, 6H), 3.34 (q, J=6.5 Hz, 1H), 3.10 (dd, J=10.5, 3.1 Hz, 1H), 1.99 (s, 3H), 1.72-1.59 (m, 1H), 1.28 (d, J=6.6 Hz, 3H), 0.96-0.77 (m, 12H), 0.13 (m, 6H).

Example 55: Synthesis of thexyldimethylsilyl 2,3,4,6-tetra-O-benzyl--D-glucopyranosyl-(14)-2-azido-2-deoxy--L-fucopyranoside (56*)

(194) ##STR00117##

(195) To a solution of disaccharide 55* (55 mg, 61 mol) in MeOH (1 mL) was added 0.5 M NaOMe in MeOH (0.12 mL) and stirred for 16 h. The mixture was neutralized with Amberlite IR 120 (H.sup.+) ion exchange resin and the resulting alcohol was filtered and concentrated. Column chromatography on silica gel (hexanes/ethyl acetate) afforded disaccharide 56* (43 mg, 50 mol, 82%). .sup.1H NMR (400 MHz, CDCl.sub.3) 7.42-7.20 (m, 18H), 7.18-7.12 (m, 2H), 5.00 (d, J=11.3 Hz, 1H), 4.85 (d, J=11.4 Hz, 2H), 4.76 (d, J=11.3 Hz, 1H), 4.63 (d, J=12.2 Hz, 1H), 4.51 (d, J=10.9 Hz, 1H), 4.48-4.32 (m, 4H), 3.93 (d, J=2.9 Hz, 1H), 3.76-3.46 (m, 7H), 3.40 (q, J=6.6 Hz, 1H), 3.18 (dd, J=10.5, 3.1 Hz, 1H), 1.73-1.63 (m, 1H), 1.32 (d, J=6.5 Hz, 3H), 0.92-0.83 (m, 12H), 0.16 (d, J=2.4 Hz, 6H).

Example 56: Synthesis of thexyldimethylsilyl [2,3,4,6-tetra-O-benzyl--D-glucopyranosyl-(14)]-3,4-di-O-benzyl-6-O-benzoyl-2-levulinoyl--D-glucopyranosyl-(13)-2-azido-2-deoxy--L-fucopyranoside (57*)

(196) ##STR00118##

(197) Acceptor 56* (40 mg, 0.047 mmol, 1.0 equiv) and 6-O-benzoyl-3,4-di-O-benzyl-2-O-levulinoyl-1-thio--D-glucopyranoside (43 mg, 0.070 mmol, 1.5 equiv) were coevaporated with toluene two times and dried in vacuo. The residue was dissolved in a mixture of toluene (2 mL) and dichloromethane (1 mL). Molecular sieves (4 ) were added and the reaction mixture cooled to 45 C. NIS (21 mg, 0.094 mmol, 2 equiv) and TfOH (0.5 L, 5.6 mol, 0.12 equiv) were added and the reaction let warm to 30 C. over two hours. The reaction was quenched with triethylamine and diluted with dichloromethane. The organic phase was washed with saturated aqueous Na.sub.2S.sub.2O.sub.3, NaHCO.sub.3 and brine. The organic layer was dried over Na.sub.2SO.sub.4 and concentrated. Column chromatography on silica gel (hexanes/ethyl acetate) afforded trisaccharide 57* (52 mg, 0.037 mmol, 79%). .sup.1H NMR (400 MHz, CDCl.sub.3) 8.10-7.98 (m, 2H), 7.57-7.50 (m, 1H), 7.47-7.09 (m, 30H), 7.07-7.00 (m, 2H), 5.23 (d, J=8.1 Hz, 1H), 5.14-5.05 (m, 1H), 4.91 (d, J=5.1 Hz, 1H), 4.88 (d, J=6.4 Hz, 1H), 4.84-4.76 (m, 2H), 4.73-4.31 (m, 11H), 4.28 (d, J=7.8 Hz, 1H), 3.99 (d, J=2.9 Hz, 1H), 3.96-3.87 (m, 2H), 3.85-3.82 (m, 1H), 3.71-3.34 (m, 8H), 3.18 (dd, J=10.4, 3.1 Hz, 1H), 2.66-2.14 (m, 4H), 2.03 (s, 1H), 1.60-1.48 (m, 1H), 1.44 (d, J=6.5 Hz, 3H), 0.83 (dd, J=7.6, 2.4 Hz, 12H), 0.12 (d, J=10.5 Hz, 6H).

Example 57: Synthesis of thexyldimethylsilyl 3-O-p-methoxybenzyl-2-azido-2-deoxy--L-fucopyranoside (59*)

(198) ##STR00119##

(199) Diol 59* (1.02 g, 3.07 mmol, 1.0 equiv) was co-evaporated with dry toluene twice and let dry in vacuo for 30 min. Then, dry toluene (30 mL) was added, followed by Bu.sub.2SnO (1.14 g, 4.60 mmol, 1.5 equiv) and 4 MS. The reaction was stirred for 1 hour under reflux. The reaction was cooled to 40 C.; PMBCl (1.25 mL, 9.20 mmol, 3 equiv) and TBAB (1.48 g, 4.60 mmol, 1.5 equiv) were added and left stir overnight at rt. The reaction mixture was filtered and the solvent evaporated. Column chromatography on silica gel (hexanes/ethyl acetate) afforded building block 59a* (1.01 g, 2.23 mmol, 73%).). [].sub.D.sup.20=25.6 (c=2.00, CHCl.sub.3); IR .sub.max (film) 3494, 2960, 2870, 2112, 1515, 1251, 1073, 829 cm.sup.1; .sup.1H NMR (400 MHz, CDCl.sub.3) 7.36-7.28 (m, 2H), 6.97-6.84 (m, 2H), 4.63 (s, 2H), 4.39 (d, J=7.7 Hz, 1H), 3.81 (s, 3H), 3.71-3.63 (m, 1H), 3.53-3.39 (m, 2H), 3.24 (dd, J=10.1, 3.3 Hz, 1H), 2.41-2.26 (m, 1H), 1.67 (hept, J=6.8 Hz, 1H), 1.31 (d, J=6.5 Hz, 3H), 0.89 (d, J=7.0 Hz, 12H), 0.17 (d, J=3.5 Hz, 6H). .sup.13C NMR (101 MHz, CDCl.sub.3) 159.6, 129.7, 129.5, 114.1, 97.0, 79.0, 71.8, 70.2, 68.4, 65.2, 55.4, 34.0, 24.9, 20.1, 20.0, 18.6, 18.5, 16.5, 1.8, 3.2. LRMS (ESI+) Calcd for C.sub.22H.sub.37N.sub.3O.sub.5SiNa.sup.+ [M+Na].sup.+ 474.2400, found 474.2.

Example 58: Synthesis of thexyldimethylsilyl 3,4,6-tri-O-benzyl-2-O-levulinoyl--D-glucopyranosyl-(14)-3-O-p-methoxybenzyl-2-azido-2-deoxy--L-fucopyranoside (61*)

(200) ##STR00120##

(201) L-fucosyl acceptor 59a* (200 mg, 0.443 mmol, 1.0 equiv) and thioglucoside 60* (354 mg, 0.70 mmol, 1.35 equiv) were coevaporated with toluene two times and dried in vacuo. The residue was dissolved in DCM (15 mL). Molecular sieves acid washed (4 ) were added and the reaction mixture cooled to 40 C. NIS (149 mg, 0.66 mmol, 1.5 equiv) and TfOH (2 L, 0.02 mmol, 0.05 equiv) were added and let stir for one hour at 40 C. The reaction was quenched with triethylamine and diluted with DCM. The organic phase was washed with saturated aqueous Na.sub.2S.sub.2O.sub.3, NaHCO.sub.3 and brine, dried over Na.sub.2SO.sub.4, filtered and concentrated. Column chromatography on silica gel (hexanes/ethyl acetate) afforded disaccharide 61* (338 mg, 0.34 mmol, 78%). [].sub.D.sup.20=2.5 (c=1.00, CHCl.sub.3); IR .sub.max (film) 2959, 2868, 2112, 1750, 1721, 1515, 1250, 1075, 832 cm.sup.1, .sup.1H NMR (600 MHz, CDCl.sub.3) 7.41-7.19 (m, 15H), 7.18-7.11 (m, 2H), 6.82 (d, J=8.4 Hz, 2H), 5.19 (t, J=8.8 Hz, 1H), 4.83-4.74 (m, 4H), 4.53-4.43 (m, 3H), 4.39 (d, J=5.3 Hz, 2H), 4.26 (d, J=7.7 Hz, 1H), 3.85-3.82 (m, 1H), 3.77 (s, 3H), 3.73-3.63 (m, 2H), 3.60-3.43 (m, 4H), 3.33 (q, J=6.6 Hz, 1H), 3.08 (dd, J=10.5, 3.0 Hz, 1H), 2.90-2.79 (m, 1H), 2.65-2.55 (m, 2H), 2.53-2.45 (m, 1H), 2.14 (s, 3H), 1.64 (h, J=6.8 Hz, 1H), 1.27 (d, J=6.6 Hz, 3H), 0.91-0.81 (m, 12H), 0.11 (d, J=12.5 Hz, 6H). .sup.13C NMR (151 MHz, CDCl.sub.3) 206.6, 171.3, 159.2, 138.4, 137.9, 130.2, 129.6, 128.6, 128.5, 128.5, 128.3, 128.2, 128.0, 128.0, 127.8, 127.8, 113.8, 101.7, 97.2, 83.2, 78.0, 75.3, 75.2, 75.0, 74.0, 73.9, 73.6, 70.3, 70.0, 69.7, 64.7, 55.4, 38.2, 34.1, 30.0, 28.0, 24.9, 20.1, 20.1, 18.63, 18.58, 17.0, 1.8, 3.1. HRMS (ESI+) Calcd for C.sub.54H.sub.71N.sub.3O.sub.12SiNa.sup.+ [M+Na].sup.+ 1004.4705, found 1004.4709.

Example 59: Synthesis of thexyldimethylsilyl 3,4,6-tri-O-benzyl--D-glucopyranosyl-(14)-3-O-p-methoxybenzyl-2-azido-2-deoxy--L-fucopyranoside (62*)

(202) ##STR00121##

(203) To a solution of disaccharide 61* (970 mg, 0.99 mmol, 1 equiv) in DCM (11 mL), a solution of hydrazine hydrate (190 L, 3.95 mmol, 4 equiv) dissolved in AcOH (1.1 mL) and pyridine (1.7 mL) was added. The resulting reaction mixture was stirred at rt for 1 to 2 h. The reaction was quenched by the addition of acetone and the solvent evaporated. Column chromatography on silica gel (hexanes/ethyl acetate) afforded disaccharide 62* (720 mg, 0.81 mmol, 82%). [].sub.D.sup.20=28.5 (c=0.10, CHCl.sub.3); IR .sub.max(film) 3470, 2930, 2870, 2113, 1515, 1252, 1115, 1069, 832 cm.sup.1, .sup.1H NMR (600 MHz, CDCl.sub.3) 7.40-7.36 (m, 2H), 7.35-7.21 (m, 13H), 7.20-7.12 (m, 2H), 6.80 (d, J=8.3 Hz, 2H), 5.00 (d, J=11.4 Hz, 1H), 4.86 (d, J=10.9 Hz, 1H), 4.80-4.72 (m, 2H), 4.60 (d, J=11.6 Hz, 1H), 4.53 (d, J=10.8 Hz, 1H), 4.50-4.40 (m, 3H), 4.36 (d, J=7.7 Hz, 1H), 3.90 (d, J=3.0 Hz, 1H), 3.74 (s, 3H), 3.69-3.44 (m, 8H), 3.39 (q, J=6.4 Hz, 1H), 3.18 (dd, J=10.5, 3.1 Hz, 1H), 1.66 (h, J=6.8 Hz, 1H), 1.29 (d, J=6.5 Hz, 3H), 0.94-0.81 (m, 12H), 0.17 (d, J=4.3 Hz, 6H). .sup.13C NMR (151 MHz, CDCl.sub.3) 159.6, 139.2, 138.3, 138.1, 130.3, 129.3, 128.51, 128.50, 128.46, 128.2, 127.94, 127.89, 127.8, 127.6, 114.0, 102.5, 97.2, 84.6, 78.0, 77.0, 75.4, 75.2, 75.1, 73.8, 73.6, 72.2, 72.1, 70.7, 69.6, 65.7, 55.4, 34.1, 25.0, 20.2, 20.1, 18.7, 18.6, 17.5, 1.7, 3.0. HRMS (ESI+) Calcd for C.sub.49H.sub.65N.sub.3O.sub.10SiNa.sup.+ [M+Na].sup.+ 906.4337, found 906.4371.

Example 60: Synthesis of thexyldimethylsilyl 2,3,4,6-tetra-O-benzyl--D-glucopyranosyl-(14)-3-O-p-methoxybenzyl-2-azido-2-deoxy--L-fucopyranoside (63*)

(204) ##STR00122##

(205) Benzyl bromide (194 L, 1.63 mmol, 2 equiv) was added to a solution of alcohol 62* (720 mg, 0.81 mmol, 1 equiv) in THF:DMF (1:1, 17 mL). Then, the mixture was cooled to 0 C. and NaH 60% in mineral oil (65 mg, 1.63 mmol, 2 equiv) was added. The reaction mixture was warmed to room temperature and let stir for 3 h. Then, it was diluted with DCM and quenched with NaHCO.sub.3. The aqueous layer was extracted with DCM (3). The organic phase was washed with brine, dried over Na.sub.2SO.sub.4, filtered and the solvent evaporated. Column chromatography on silica gel (hexanes/ethyl acetate) afforded disaccharide 63* (715 mg, 0.73 mmol, 90%). [].sub.D.sup.20=5.4 (c=2.44, CHCl.sub.3); IR .sub.max (film) 3066, 3033, 2957, 2867, 2112, 1514, 1249, 1068, 831, 698 cm.sup.1, .sup.1H NMR (400 MHz, CDCl.sub.3) 7.47-7.07 (m, 22H), 6.83 (d, J=8.2 Hz, 2H), 5.17 (d, J=10.4 Hz, 1H), 5.04 (d, J=11.1 Hz, 1H), 4.90-4.69 (m, J=25.2, 21.0, 11.5 Hz, 5H), 4.54-4.32 (m, 6H), 3.97 (d, J=3.2 Hz, 1H), 3.78 (s, 3H), 3.73-3.36 (m, 9H), 3.15 (dd, J=10.5, 3.1 Hz, 1H), 1.68-1.56 (h, J=6.8 Hz, 1H), 1.38 (d, J=6.5 Hz, 3H), 0.92-0.78 (m, 12H), 0.15 (s, 6H). .sup.13C NMR (101 MHz, CDCl.sub.3) 159.2, 138.9, 138.2, 137.9, 137.8, 130.1, 129.8, 129.3, 128.52, 128.49, 128.47, 128.34, 128.32, 128.03, 127.97, 127.8, 127.7, 113.7, 104.5, 97.1, 85.0, 81.8, 77.9, 76.6, 75.8, 75.1, 75.0, 74.9, 73.7, 73.5, 70.5, 70.0, 69.6, 65.2, 55.3, 34.0, 24.9, 20.2, 20.0, 18.6, 18.5, 17.3, 1.9, 3.2. HRMS (ESI+) Calcd for C.sub.56H.sub.71N.sub.3O.sub.10SiNa.sup.+ [M+Na].sup.+ 1004.4806, found 996.4809.

Example 61: Synthesis of thexyldimethylsilyl 2,3,4,6-tetra-O-benzyl--D-glucopyranosyl-(14)-2-azido-2-deoxy--L-fucopyranoside (64*)

(206) ##STR00123##

(207) To a solution of disaccharide 63* (630 mg, 0.65 mmol, 1 equiv) in DCM:H.sub.2O (18:1, 5.3 mL) was added 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) (161 mg, 0.71 mmol, 1.1 equiv) at 0 C. The reaction was warmed to rt and let stir for 2.5 h. The reaction was not complete, then 10 mg DDQ were added. After 30 minutes, the reaction was diluted with DCM and NaHCO.sub.3 added. The organic phase was washed (3) with NaHCO.sub.3 until the solution was colorless. Then, the organic phase was washed with brine, dried over Na.sub.2SO.sub.4, filtered and the solvent evaporated. Column chromatography on silica gel (hexanes/ethyl acetate) afforded disaccharide 64* (460 mg, 0.54 mmol, 83%). [].sub.D.sup.20=1.9 (c=1.36, CHCl.sub.3); IR .sub.max (film) 3484, 3034, 2959, 2869, 2112, 1254, 1069, 833, 629 cm.sup.1, .sup.1H NMR (400 MHz, CDCl.sub.3) 7.43-7.19 (m, 18H), 7.16-7.08 (m, 2H), 5.08-4.97 (m, 2H), 4.87-4.75 (m, 3H), 4.59-4.44 (m, 4H), 4.37 (d, J=7.7 Hz, 1H), 3.73-3.34 (m, 10H), 1.66 (h, J=7.0 Hz, 1H), 1.30 (d, J=6.4 Hz, 3H), 0.93-0.78 (m, 12H), 0.20 (s, 6H). .sup.13C NMR (101 MHz, CDCl.sub.3) 138.6, 137.9, 137.85, 137.81, 128.8, 128.6, 128.5, 128.4, 128.2, 128.1, 128.0, 127.9, 127.9, 127.8, 104.0, 97.3, 84.6, 81.5, 80.6, 77.6, 77.4, 75.9, 75.3, 75.0, 73.8, 71.4, 70.3, 68.8, 67.7, 34.1, 24.9, 20.2, 20.0, 18.6, 18.5, 16.7, 1.9, 3.0. HRMS (ESI+) Calcd for C.sub.48H.sub.63N.sub.3O.sub.9SiNa.sup.+ [M+Na].sup.+ 876.4231, found 876.4234.

Example 62: Synthesis of thexyldimethylsilyl [2,3,4,6-tetra-O-benzyl--D-glucopyranosyl-(14)]-benzyl-3,4-di-O-benzyl-6-O-benzoyl-2-levulinoyl--D-glucopyranosyluronate-(13)-2-azido-2-deoxy--L-fucopyranoside (66*)

(208) ##STR00124##

(209) Acceptor 64* (91 mg, 0.11 mmol, 1.0 equiv) and thioglucoside 65* (129 mg, 0.21 mmol, 2 equiv) were coevaporated with toluene two times and dried in vacuo. The residue was dissolved in a mixture of toluene (4 mL) and DCM (2 mL). Molecular sieves acid washed (4 ) were added and the reaction mixture cooled to 30 C. NIS (53 mg, 0.23 mmol, 2.2 equiv) and TfOH (0.5 L, 5.3 mol, 0.05 equiv) were added and the reaction let warm to 15 C. over two hours. The reaction was quenched with triethylamine and diluted with DCM. The organic phase was washed with saturated aqueous Na.sub.2S.sub.2O.sub.3, NaHCO.sub.3 and brine, dried over Na.sub.2SO.sub.4, filtered and concentrated. Column chromatography on silica gel (hexanes/ethyl acetate) afforded trisaccharide 66* (114 mg, 0.08 mmol, 76%). [].sub.D.sup.20=26.6 (c=0.87, CHCl.sub.3); IR .sub.max (film) 3034, 2929, 2870, 2116, 1751, 1720, 1072, 832, 698 cm.sup.1, .sup.1H NMR (400 MHz, CDCl.sub.3) 7.56-6.90 (m, 35H), 5.21-5.06 (m, 5H), 5.02-4.94 (m, 2H), 4.83-4.61 (m, 6H), 4.53-4.32 (m, 6H), 4.06-3.88 (m, 3H), 3.83-3.73 (m, 2H), 3.65-3.33 (m, 8H), 3.21 (t, J=9.2 Hz, 1H), 2.78-2.57 (m, 2H), 2.50-2.37 (m, 2H), 2.11 (s, 3H), 1.68-1.53 (m, 1H), 1.35 (d, J=6.5 Hz, 3H), 0.94-0.73 (m, 12H), 0.15 (d, J=8.8 Hz, 6H). .sup.13C NMR (101 MHz, CDCl.sub.3) 206.3, 171.3, 168.6, 138.8, 138.4, 138.3, 138.2, 138.0, 137.9, 135.3, 129.2, 128.7, 128.65, 128.60, 128.56, 128.55, 128.53, 128.4, 128.20, 128.18, 128.1, 128.0, 127.9, 127.8, 127.73, 127.70, 127.67, 104.0, 96.8, 95.5, 84.9, 82.0, 81.4, 79.5, 78.3, 75.9, 75.2, 74.9, 74.8, 74.4, 74.3, 74.1, 73.4, 73.4, 73.0, 72.4, 70.5, 69.2, 67.3, 64.6, 37.9, 34.1, 30.1, 28.2, 24.9, 20.2, 20.0, 18.7, 18.5, 17.5, 1.9, 3.2. HRMS (ESI+) Calcd for C.sub.80H.sub.95N.sub.3O.sub.17SiNa.sup.+ [M+Na].sup.+ 1420.6328, found 1420.6322.

Example 63: Synthesis of [2,3,4,6-Tetra-O-benzyl--D-glucopyranosyl-(14)]-benzyl-3,4-di-O-benzyl-6-O-benzoyl-2-levulinoyl--D-glucopyranosyluronate-(13)-2-azido-2-deoxy-,-L-fucopyranoside (67*)

(210) ##STR00125##

(211) Trisaccharide 66* (235 mg, 0.17 mmol, 1.0 equiv) was dissolved in a mixture of pyridine (1 mL) and DCM (0.5 mL) in a plastic falcon tube. HF.py (0.15 mL, 1.70 mmol, 10 equiv) was added dropwise and the reaction let stir at rt for 48-72 h. The reaction was diluted with DCM and quenched with NaHCO.sub.3. The phases were separated and the organic phase washed with brine, dried over Na.sub.2SO.sub.4, filtered and concentrated. Column chromatography on silica gel (hexanes/ethyl acetate) afforded trisaccharide lactol 67* (180 mg, 0.14 mmol, 85%). : (1.4:1) mixture: clear oil. [].sub.D.sup.20=36.8 (c=1.12, CHCl.sub.3); IR .sub.max (film) 3480, 3033, 2912, 2870, 2115, 1750, 1720, 1071, 740, 698 cm.sup.1, .sup.1H NMR (600 MHz, CDCl.sub.3) 7.48-7.03 (m, 60H), 5.29 (d, J=3.5 Hz, 1H), 5.21-5.05 (m, 9H), 5.00-4.94 (m, 2H), 4.82-4.76 (m, 4H), 4.74-4.63 (m, 7H), 4.53-4.43 (m, 9H), 4.35 (dd, J=10.2, 3.0 Hz, 1H), 4.16 (q, J=6.7 Hz, 1H), 4.07 (d, J=2.9 Hz, 1H), 4.03-3.95 (m, 4H), 3.90 (dd, J=10.2, 3.5 Hz, 1H), 3.87 (dd, J=10.4, 3.1 Hz, 1H), 3.84-3.77 (m, 2H), 3.67-3.59 (m, 4H), 3.57-3.48 (m, 3H), 3.47-3.38 (m, 3H), 3.31-3.25 (m, 2H), 2.70-2.65 (m, 3H), 2.56-2.47 (m, 2H), 2.45-2.36 (m, 2H), 2.13 (s, 2H), 2.11 (s, 3H), 1.39 (d, J=6.6 Hz, 2H), 1.36 (d, J=6.7 Hz, 3H). .sup.13C NMR (151 MHz, CDCl.sub.3) 206.4, 206.1, 171.3, 168.5, 168.4, 138.7, 138.40, 138.38, 138.3, 138.1, 138.00, 137.98, 135.3, 135.2, 129.0, 128.9, 128.73, 128.72, 128.67, 128.64, 128.61, 128.58, 128.54, 128.53, 128.50, 128.47, 128.39, 128.37, 128.19, 128.16, 128.1, 128.05, 127.98, 127.93, 127.92, 127.88, 127.86, 127.84, 127.77, 127.75, 127.71, 103.8, 103.7, 96.14, 96.09, 96.0, 91.9, 84.9, 82.6, 82.4, 81.44, 81.40, 79.7, 79.5, 78.3, 75.9, 75.2, 75.14, 75.13, 75.06, 74.96, 74.94, 74.6, 74.5, 74.43, 74.40, 74.3, 74.2, 73.6, 73.4, 73.3, 73.0, 72.3, 71.7, 71.0, 69.2, 69.1, 67.4, 67.1, 63.6, 59.9, 37.8, 30.10, 30.07, 28.3, 17.5, 17.2. HRMS (ESI+) Calcd for C.sub.72H.sub.77N.sub.3O.sub.17Na.sup.+ [M+Na].sup.+ 1278.5151, found 1278.5177.

Example 64: Synthesis of N-(Benzyl)benzyloxycarbonyl-5-amino-pentanyl [2,3,4,6-tetra-O-benzyl--D-glucopyranosyl-(14)]-benzyl-3,4-di-O-benzyl-6-O-benzoyl-2-levulinoyl--D-glucopyranosyluronate-(13)-2-azido-2-deoxy--L-fucopyranosyl-(13)-2-azido-2-deoxy-4-O-(2-naphthalenylmethyl)--D-fucopyranoside (69*)

(212) ##STR00126##

(213) To a solution of the trisaccharide lactol 67* (80 mg, 0.06 mmol, 1.0 equiv) in DCM (3 mL) were added Cs.sub.2CO.sub.3 (42 mg, 0.12 mmol, 2.0 equiv) and 2,2,2-trifluoro-N-phenylacetimidoyl chloride (30 L, 0.19 mmol, 3.0 equiv) at 0 C. After 2 h at rt, the product (68*) was filtered and the solvent evaporated. The crude mixture was passed through a small plug of silica and used in the next reaction without further purification. Acceptor 6* (81 mg, 0.13 mmol, 2.0 equiv) and trisaccharide imidate 68* (90 mg, 0.06 mmol, 1.0 equiv) were coevaporated with toluene two times and dried in vacuo. The residue was dissolved in DCM (3 mL). Molecular sieves acid washed (4 ) were added and the reaction mixture cooled to 35 C. TMSOTf (1 L, 6 mol, 0.1 equiv) was added and the reaction let warm to 20 C. over 1 h. The reaction was quenched with triethylamine, filtered and the solvent evaporated. Column chromatography on silica gel (hexanes/ethyl acetate) afforded tetrasaccharide 69* (94 mg, 0.05 mmol, 79%). [].sub.D.sup.20=52.6 (c=0.54, CHCl.sub.3); IR .sub.max (film) 3033, 2929, 2870, 2116, 1751, 1699, 1361, 1071, 737, 698 cm.sup.1, .sup.1H NMR (600 MHz, CDCl.sub.3) 7.81-7.75 (m, 3H), 7.72 (s, 1H), 7.51-7.48 (m, 1H), 7.45-7.40 (m, 2H), 7.39-7.07 (m, 43H), 7.04 (d, J=7.5 Hz, 2H), 5.26 (d, J=3.6 Hz, 1H), 5.20-5.14 (m, 2H), 5.12-5.05 (m, 2H), 4.99-4.94 (m, 2H), 4.91 (d, J=11.0 Hz, 1H), 4.86 (d, J=12.2 Hz, 1H), 4.83-4.71 (m, 4H), 4.69-4.59 (m, 4H), 4.54-4.40 (m, 6H), 4.35 (d, J=7.9 Hz, 1H), 4.30 (dd, J=10.3, 3.0 Hz, 1H), 4.22-4.12 (m, 1H), 4.03 (d, J=9.9 Hz, 1H), 3.95 (t, J=9.3 Hz, 1H), 3.88-3.82 (m, 3H), 3.79-3.73 (m, 1H), 3.73-3.65 (m, 2H), 3.63-3.54 (m, 2H), 3.53-3.35 (m, 7H), 3.32 (t, J=9.4 Hz, 1H), 3.29-3.23 (m, 1H), 3.22-3.16 (m, 1H), 2.63 (t, J=6.6 Hz, 2H), 2.44 (dt, J=17.4, 6.5 Hz, 1H), 2.36 (dt, J=17.4, 6.7 Hz, 1H), 2.08 (s, 3H), 1.68-1.46 (m, 4H), 1.43-1.27 (m, 2H), 1.31 (d, J=6.4 Hz, 3H), 1.00 (d, J=6.5 Hz, 3H). .sup.13C NMR (151 MHz, CDCl.sub.3) 206.2, 171.3, 168.1, 138.7, 138.4, 138.2, 138.0, 135.8, 135.1, 133.2, 133.1, 128.7, 128.62, 128.59, 128.56, 128.55, 128.52, 128.51, 128.46, 128.4, 128.3, 128.21, 128.20, 128.1, 128.03, 127.99, 127.97, 127.9, 127.82, 127.75, 127.7, 127.5, 127.4, 127.1, 126.3, 126.2, 103.7, 102.74, 99.1, 97.0, 85.0, 82.4, 81.4, 79.7, 79.0, 78.3, 75.8, 75.2, 74.9, 74.8, 74.72, 74.68, 74.4, 73.7, 73.2, 71.0, 69.2, 67.3, 67.2, 64.0, 59.0, 50.7, 50.4, 47.3, 46.4, 37.8, 30.1, 29.4, 28.2, 23.4, 17.4, 16.8. HRMS (ESI+) Calcd for C.sub.109H.sub.117N.sub.7O.sub.22Na.sup.+ [M+Na].sup.+ 1900.1187, found 1900.184.

Example 65: Synthesis of N-(Benzyl)benzyloxycarbonyl-5-amino-pentanyl [2,3,4,6-tetra-O-benzyl--D-glucopyranosyl-(14)]-benzyl-3,4-di-O-benzyl-6-O-benzoyl--D-glucopyranosyluronate-(13)-2-azido-2-deoxy--L-fucopyranosyl-(13)-2-azido-2-deoxy-4-O-(2-naphthalenylmethyl)--D-fucopyranoside (70*)

(214) ##STR00127##

(215) To a solution of tetrasaccharide 69* (40 mg, 0.021 mmol, 1 equiv) in DCM (2 mL), a solution of hydrazine hydrate (13 L, 0.26 mmol, 12 equiv) dissolved in AcOH (0.08 mL) and pyridine (0.12 mL) was added. The resulting reaction mixture was stirred at rt for 1 to 2 h. The reaction was quenched by the addition of acetone and the solvent evaporated. Column chromatography on silica gel (hexanes/ethyl acetate) afforded tetrasaccharide 70* (34 mg, 0.019 mmol, 90%). [].sub.D20=66.6 (c=1.00, CHCl.sub.3); IR .sub.max (film) 3501, 3033, 2928, 2870, 2116, 1749, 1698, 1070, 735, 698 cm.sup.1, .sup.1H NMR (400 MHz, CDCl.sub.3) 7.87-7.74 (m, 3H), 7.17 (s, 1H), 7.58-6.92 (m, 48H), 5.29 (d, J=3.7 Hz, 1H), 5.21-5.13 (m, 3H), 5.08-4.71 (m, 10H), 4.59-4.38 (m, 6H), 4.37-4.29 (m, 2H), 4.27-4.18 (m, 2H), 4.00-3.86 (m, 5H), 3.78-3.17 (m, 17H), 1.72-1.47 (m, 4H), 1.39 (d, J=6.4 Hz, 3H), 1.43-1.27 (m, 2H), 1.04 (d, J=6.6 Hz, 3H). .sup.13C NMR (101 MHz, CDCl.sub.3) 168.0, 156.8, 156.3, 138.7, 138.7, 138.2, 138.1, 138.0, 137.83, 137.78, 135.6, 135.3, 133.1, 133.0, 128.9, 128.7, 128.62, 128.61, 128.51, 128.49, 128.47, 128.44, 128.40, 128.35, 128.26, 128.2, 128.1, 127.92, 127.89, 127.86, 127.8, 127.7, 127.6, 127.3, 127.0, 126.7, 126.5, 126.3, 104.1, 103.4, 102.8, 100.7, 84.6, 83.9, 82.3, 79.0, 78.8, 78.2, 77.6, 75.8, 75.7, 75.3, 75.23, 75.20, 75.1, 75.0, 74.2, 73.2, 71.0, 70.0, 69.9, 68.2, 67.4, 67.2, 66.8, 63.8, 58.0, 50.6, 50.3, 47.2, 46.3, 29.3, 28.0, 27.5, 23.3, 17.5, 16.8. HRMS (ESI+) Calcd for C.sub.104H.sub.111N.sub.7O.sub.20Na.sup.+ [M+Na].sup.+ 1801.7815, found 1801.7992.

Example 66: Synthesis of N-(Benzyl)benzyloxycarbonyl-5-amino-pentanyl 2-azido-3,4-di-O-benzyl-2-deoxy--L-pneumopyranosyl-(12)-[2,3,4,6-tetra-O-benzyl--D-glucopyranosyl-(14)]-benzyl-3,4-di-O-benzyl-6-O-benzoyl--D-glucopyranosyluronate-(13)-2-azido-2-deoxy--L-fucopyranosyl-(13)-2-azido-2-deoxy-4-O-(2-naphthalenylmethyl)--D-fucopyranoside (71*)

(216) ##STR00128##

(217) Acceptor 70* (25 mg, 0.014 mmol, 1.0 equiv) and imidate 8a* (23 mg, 0.042 mmol, 3.0 equiv) were coevaporated with toluene two times and dried in vacuo. The residue was dissolved in toluene (2 mL). The reaction mixture cooled to 35 C. TMSOTf (0.25 L, 1.4 mol, 0.1 equiv) was added and the reaction let warm to 20 C. over 2 h. The reaction was quenched with triethylamine, filtered and the solvent evaporated. Column chromatography on silica gel (hexanes/ethyl acetate) afforded pentasaccharide 71* (16 mg, 0.07 mmol, 53%). [].sub.D.sup.20=39.5 (c=0.30, CHCl.sub.3); IR .sub.max (film) 3034, 2931, 2870, 2115, 1750, 1698, 1455, 1070, 736, 698 cm.sup.1, .sup.1H NMR (600 MHz, CDCl.sub.3) 7.87-7.73 (m, 3H), 7.68 (s, 1H), 7.62-7.02 (m, 55H), 7.02-6.86 (m, 3H), 5.32 (d, J=3.9 Hz, 1H), 5.28-5.22 (m, 2H), 5.21-5.13 (m, 2H), 5.03-4.39 (m, 22H), 4.33 (d, J=7.9 Hz, 1H), 4.26 (d, J=10.9 Hz, 1H), 4.24-4.16 (m, 2H), 4.06 (d, J=9.7 Hz, 1H), 4.01-3.86 (m, 4H), 3.85-3.79 (m, 2H), 3.73 (t, J=8.3 Hz, 1H), 3.67-3.54 (m, 4H), 3.54-3.32 (m, 8H), 3.31-3.12 (m, 4H), 1.74-1.46 (m, 4H), 1.46-1.18 (m, 8H), 0.97 (d, J=6.7 Hz, 3H). .sup.13C NMR (151 MHz, CDCl.sub.3) 168.1, 138.7, 138.4, 137.7, 137.60, 135.58, 134.9, 133.2, 128.73, 128.65, 128.62, 128.57, 128.5, 128.44, 128.42, 128.37, 128.35, 128.23, 128.20, 128.1, 128.03, 127.97, 127.91, 127.87, 127.8, 127.7, 127.6, 127.5, 127.4, 127.2, 127.1, 126.7, 126.4, 126.2, 103.9, 102.9, 100.3, 99.2, 95.4, 85.2, 84.1, 81.2, 80.4, 78.8, 78.2, 77.5, 76.8, 76.5, 75.6, 75.1, 74.9, 74.84, 74.77, 74.5, 74.2, 73.2, 72.5, 71.1, 70.5, 70.0, 69.92, 69.87, 69.3, 68.0, 67.3, 67.0, 66.9, 66.5, 64.0, 57.9, 57.3, 50.7, 50.4, 47.3, 47.3, 46.3, 32.1, 29.8, 29.5, 29.4, 28.0, 27.60, 23.4, 23.3, 22.8, 17.5, 17.1, 17.0, 14.7, 14.3. HRMS (ESI+) Calcd for C.sub.124H.sub.132N.sub.10O.sub.23Na.sup.+ [M+Na].sup.+ 2152.9398, found 2152.9582.

Example 67: Synthesis of N-(Benzyl)benzyloxycarbonyl-5-amino-pentanyl 2-N-acetyl-3,4-di-O-benzyl--L-pneumopyranosyl-(12)-[2,3,4,6-tetra-O-benzyl--D-glucopyranosyl-(14)]-benzyl-3,4-di-O-benzyl-6-O-benzoyl--D-glucopyranosyluronate-(13)-2-N-acetyl--L-fucopyranosyl-(13)-2-N-acetyl-4-O-(2-naphthalenylmethyl)--D-fucopyranoside (72*)

(218) ##STR00129##

(219) To a solution of azido pentasaccharide 71* (32 mg, 0.015 mmol, 1 equiv) in pyridine (2 mL), thioacetic acid (0.5 mL) was added and stirred for 5 days at rt. The reaction mixture was concentrated and purified by column chromatography on silica gel (hexanes/acetone) to afford the acetamide pentasaccharide 72* (32 mg, 0.015 mmol, 98%). [].sub.D.sup.20=26.6 (c=0.87, CHCl.sub.3); IR .sub.max (film) 3420, 3330, 3030, 2935, 2870, 1677, 1455, 1364, 1070, 7, 698 cm.sup.1, .sup.1H NMR (400 MHz, CDCl.sub.3) 7.92-7.67 (m, 5H), 7.63-6.87 (m, 55H), 6.80 (d, J=7.5 Hz, 2H), 6.09 (bs, 1H), 5.76 (bs, 1H), 5.51 (bs, 1H), 5.46-5.36 (m, 2H), 5.28 (s, 1H), 5.22-5.12 (m, 2H), 5.07-4.96 (m, 3H), 4.93-4.33 (m, 22H), 4.29-4.08 (m, 5H), 3.96-3.58 (m, 11H), 3.56-3.30 (m, 6H), 3.28-3.16 (m, 3H), 2.03-1.85 (m, 6H), 1.65-1.45 (m, 7H), 1.36-1.08 (m, 11H). .sup.13C NMR (151 MHz, CDCl.sub.3) 171.6, 170.7, 169.7, 169.4, 138.6, 138.3, 138.2, 138.02, 137.98, 137.8, 137.7, 137.6, 136.3, 134.5, 133.2, 133.1, 128.9, 128.7, 128.65, 128.62, 128.58, 128.48, 128.43, 128.36, 128.33, 128.29, 128.2, 128.1, 128.02, 127.97, 127.94, 127.89, 127.69, 127.67, 127.64, 127.56, 127.44, 127.39, 126.9, 126.53, 126.49, 126.2, 103.8, 100.5, 99.7, 97.0, 95.0, 85.3, 84.5, 80.9, 80.6, 78.8, 78.3, 77.8, 76.2, 75.8, 75.3, 75.1, 74.4, 74.22, 73.4, 73.2, 71.7, 70.7, 69.8, 69.5, 68.3, 67.4, 67.3, 66.6, 66.5, 55.2, 50.6, 50.4, 49.6, 48.0, 47.4, 46.2, 29.3, 27.99, 27.5, 24.0, 23.6, 23.3, 17.6, 17.5, 16.5. HRMS (ESI+) Calcd for C.sub.130H.sub.144N.sub.4O.sub.26Na.sup.+ [M+Na].sup.+ 2201.0000, found 2201.0086.

Example 68: Synthesis of 5-amino-pentanyl 2-N-acetyl--L-pneumopyranosyl-(12)-[-D-glucopyranosyl-(14)]--D-glucopyranosyluronate-(13)-2-N-acetyl--L-fucopyranosyl-(13)-2-N-acetyl--D-fucopyranoside (73*)

(220) ##STR00130##

(221) The pentasaccharide 72* was dissolved in CH.sub.2Cl.sub.2/tBuOH/water (1.5:16:8, 1 mL, purged with argon and treated with a suspension of Pd(OH).sub.2 on carbon (20% (w/w) loading, 10 mg) in the same solvent mixture (1 mL). The suspension was purged with hydrogen, stirred under hydrogen atmosphere at rt. After 24 h, a freshly prepared suspension of Pd(OH).sub.2 (5 mg, 1 mL) was added and the reaction was stirred for 24 h more, filtered and concentrated. The residue was purified by solid phase extraction (Chromabond C18, Macherey-Nagel) and size exclusion chromatography on Sephadex LH-20 (MeOH:H.sub.2O) and lyophilized to give pentasaccharide 73* (3 mg, 2.99 mol, 50%) as a white solid.

(222) .sup.1H NMR (400 MHz, D.sub.2O) 5.16 (d, J=4.0 Hz, 1H), 5.07 (s, 1H), 5.00 (d, J=7.6 Hz, 1H), 4.58-4.47 (m, 2H), 4.44-4.28 (m, 3H), 4.17-4.05 (m, 3H), 4.00-3.61 (m, 12H), 3.56-3.27 (m, 7H), 3.11 (dd, J=9.3, 7.9 Hz, 1H), 2.99-2.88 (m, 2H), 2.00 (s, 3H), 1.96 (s, 3H), 1.95 (s, 3H), 1.69-1.46 (m, 4H), 1.41-1.26 (m, 5H), 1.22 (d, J=6.4 Hz, 3H), 1.05 (d, J=6.5 Hz, 3H). .sup.13C NMR (101 MHz, D.sub.2O) 176.0, 174.01, 173.97, 173.3, 102.8, 101.6, 99.7, 97.3, 93.7, 77.8, 76.0, 75.8, 75.2, 73.8, 71.9, 71.7, 71.0, 70.9, 70.6, 70.3, 69.9, 69.8, 67.0, 66.9, 63.8, 60.6, 51.4, 50.8, 49.0, 39.2, 28.1, 26.3, 22.3, 22.1, 21.9, 15.9, 15.3, 15.2. HRMS (ESI+) Calcd for C.sub.141H.sub.70N.sub.4O.sub.24Na.sup.+ [M+Na].sup.+ 1026. 4311, found 1026.4397.

Example 69: N-(Benzyl)-benzyloxycarbonyl-5-amino-pentanyl 2-azido-2-deoxy-4-O-(2-naphthalenylmethyl)-3-O-allyl--D-fucopyranoside (74*)

(223) ##STR00131##

(224) Allyl bromide (30 L, 0.35 mmol, 1.5 equiv) was added to a solution of alcohol 6* (150 mg, 0.24 mmol, 1 equiv) in THF:DMF (9:1, 2.0 mL). Then, the mixture was cooled to 0 C. and NaH 60% in mineral oil (19 mg, 0.47 mmol, 2 equiv) was added. The reaction was warmed to room temperature and let stir overnight. The reaction mixture was diluted with DCM and quenched with NaHCO.sub.3. The organic phase was washed with brine, dried over Na.sub.2SO.sub.4, filtered and the solvent evaporated. Column chromatography on silica gel (hexanes/ethyl acetate) afforded the allyl fucopyranoside 74* (145 mg, 0.21 mmol, 91%). .sup.1H NMR (600 MHz, CDCl.sub.3) 7.41-7.19 (m, 15H), 7.18-7.11 (m, 2H), 6.82 (d, J=8.4 Hz, 2H), 5.19 (t, J=8.8 Hz, 1H), 4.83-4.74 (m, 4H), 4.53-4.43 (m, 3H), 4.39 (d, J=5.3 Hz, 2H), 4.26 (d, J=7.7 Hz, 1H), 3.85-3.82 (m, 1H), 3.77 (s, 3H), 3.73-3.63 (m, 2H), 3.60-3.43 (m, 4H), 3.33 (q, J=6.6 Hz, 1H), 3.08 (dd, J=10.5, 3.0 Hz, 1H), 2.90-2.79 (m, 1H), 2.65-2.55 (m, 2H), 2.53-2.45 (m, 1H), 2.14 (s, 3H), 1.64 (h, J=6.8 Hz, 1H), 1.27 (d, J=6.6 Hz, 3H), 0.91-0.81 (m, 12H), 0.11 (d, J=12.5 Hz, 6H). .sup.13C NMR (151 MHz, CDCl.sub.3) 156.8, 156.3, 138.0, 137.0, 136.9, 135.8, 134.5, 133.2, 133.1, 128.6, 128.5, 128.1, 128.0, 127.9, 127.8, 127.3, 127.2, 126.7, 126.1, 126.0, 117.6, 102.3, 81.0, 74.7, 71.6, 70.6, 69.7, 69.6, 67.2, 63.0, 50.6, 50.3, 47.2, 46.3, 29.3, 28.0, 27.5, 23.3, 17.1. HRMS (ESI+) Calcd for C.sub.40H.sub.46N.sub.4O.sub.6Na.sup.+ [M+Na].sup.+ 701.3315, found 701.3336.

Example 70: N-(Benzyl)-benzyloxycarbonyl-5-amino-pentanyl 2-azido-2-deoxy-4-O-acetyl-3-O-allyl--D-quinovopyranoside (77*)

(225) ##STR00132##

(226) To a solution of fucopyranoside 74* (145 mg, 0.21 mmol, 1 equiv) in DCM:H.sub.2O (18:1, 2 mL) was added 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (58 mg, 0.26 mmol, 1.2 equiv) at 0 C. The reaction was warmed to rt and let stir for 2.5 h. The reaction was diluted with DCM and NaHCO.sub.3 was added. The organic phase was washed (3) with NaHCO.sub.3 until the solution was colorless. Then, the organic phase was washed with brine, dried over Na.sub.2SO.sub.4 and the solvent evaporated. Column chromatography on silica gel (hexanes/ethyl acetate) afforded fucopyranoside 75* (70 mg, 0.13 mmol, 61%). To a solution of fucopyranoside 75* (35 mg, 0.065 mmol, 1 equiv) in DCM (1 mL) was added pyridine (32 L, 0.390 mmol, 6 equiv) and triflic anhydride(22 L, 0.130 mmol, 2 equiv) at 0 C. The resulting deep purple colored reaction mixture was let stir at 00 C. for 30 min. Then, the reaction was diluted with DCM and quenched with NaHCO.sub.3. The phases were separated and the organic layer was washed with brine, dried over Na.sub.2SO.sub.4, filtered and the solvent evaporated. The resulting oil was co-evaporated with toluene, dissolved in DMF (1 mL) and used in the next step without further purification. Tetrabutylammonium acetate was added to the solution of the triflate (76*) in DMF and let stir overnight at rt. The solvent was evaporated. Column chromatography on silica gel (hexanes/ethyl acetate) afforded the quinovopyranoside 77* (26 mg, 0.044 mmol, 68%). [].sub.D.sup.20=19.6 (c=0.56, CHCl.sub.3); IR .sub.max (film) 2939, 2868, 2111, 1747, 1698, 1423, 1230, 1066, 699 cm.sup.1; .sup.1H NMR (400 MHz, CDCl.sub.3) 7.45-7.08 (m, 10H), 5.86 (ddt, J=17.2, 10.4, 5.7 Hz, 1H), 5.30-5.10 (m, 4H), 4.74 (t, J=9.5 Hz, 1H), 4.56-4.41 (m, 2H), 4.32-4.15 (m, 2H), 4.12-4.05 (m, 1H), 3.94-3.76 (m, 1H), 3.56-3.32 (m, 3H), 3.30-3.16 (m, 3H), 2.09 (s, 3H), 1.69-1.46 (m, 4H), 1.42-1.26 (m, 2H), 1.19 (d, J=6.2 Hz, 3H). .sup.13C NMR (151 MHz, CDCl.sub.3) 169.8, 138.1, 134.6, 128.7, 128.6, 128.04, 127.97, 127.4, 117.3, 102.0, 80.1, 74.9, 73.7, 70.2, 67.3, 66.2, 50.7, 50.4, 47.2, 46.3, 29.3, 28.0, 27.6, 23.3, 21.1, 17.5. HRMS (ESI+) Calcd for C.sub.31H.sub.40N.sub.4O.sub.7Na.sup.+ [M+Na].sup.+ 603.2795, found 603.2809.

Example 71: N-(Benzyl)-benzyloxycarbonyl-5-amino-pentanyl 2-azido-2-deoxy-4-O-(2-naphthalenylmethyl)-3-O-allyl--D-quinovopyranoside (79*)

(227) ##STR00133##

(228) To a solution of quinovopyranoside 77* (35 mg, 0.060 mmol, 1 equiv) in MeOH (0.5 mL) was added 0.5 M NaOMe in MeOH (0.362 mL, 0.181 mmol, 3 equiv) and stirred overnight. The mixture was neutralized with Amberlite IR 120 (H.sup.+) ion exchange resin, filtered and concentrated in vacuo. Column chromatography on silica gel (hexanes/ethyl acetate) afforded alcohol (78*) (32 mg, 0.059 mmol, 99%). 2-naphthalenylmethyl bromide (20 mg, 0.091 mmol, 1.5 equiv) was added to a solution of alcohol (78*) (32.5 mg, 0.060 mmol, 1 equiv) in THF: DMF (9:1, 1.0 mL). Then, the mixture was cooled to 0 C. and NaH 60% in mineral oil (3 mg, 0.121 mmol, 2 equiv) was added. The reaction was warmed to room temperature. After 1.5 h, 2 equiv of NaH were added and the reaction let stir for further 1 h. The reaction mixture was diluted with DCM and quenched with NaHCO.sub.3. Then, the organic phase was washed with brine, dried over Na.sub.2SO.sub.4, filtered and the solvent evaporated. Column chromatography on silica gel (hexanes/ethyl acetate) afforded the quinovopyranoside 79* (35 mg, 0.052 mmol, 82%). .sup.1H NMR (400 MHz, CDCl.sub.3) 7.78-7.71 (m, 3H), 7.70 (bs, 1H), 7.45-7.33 (m, 3H), 7.33-7.04 (m, 10H), 5.92 (ddt, J=16.4, 10.3, 5.8 Hz, 1H), 5.24 (dd, J=16.0, 1.6 Hz, 1H), 5.18-5.03 (m, 3H), 4.83 (ABq, J=11.0 Hz, 2H), 4.42 (d, J=7.7 Hz, 2H), 4.27 (qdt, J=12.2, 5.9, 1.5 Hz, 2H), 4.11 (t, J=8.7 Hz, 1H), 3.83-3.69 (tt, J=15.6, 6.3 Hz, 1H), 3.47-3.03 (m, 7H), 1.62-1.37 (m, 4H), 1.35-1.16 (m, 2H), 1.22 (d, J=6.1 Hz, 3H). .sup.13C NMR (101 MHz, CDCl.sub.3) 156.8, 156.3, 138.0, 137.0, 136.9, 135.5, 134.8, 133.4, 133.1, 128.63, 128.57, 128.4, 128.0, 127.9, 127.8, 127.4, 127.3, 126.9, 126.3, 126.2, 126.1, 101.9, 83.3, 82.7, 75.5, 74.4, 71.4, 70.0, 69.9, 67.2, 66.5, 50.6, 50.3, 47.2, 46.3, 29.3, 28.0, 27.5, 23.3, 18.0. HRMS (ESI+) Calcd for C.sub.40H.sub.46N.sub.4O.sub.6Na.sup.+ [M+Na].sup.+ 701.3315, found 701.3348.

Example 72: N-(Benzyl)-benzyloxycarbonyl-5-amino-pentanyl 2-azido-2-deoxy-4-O-(2-naphthalenylmethyl)--D-quinovopyranoside (80*)

(229) ##STR00134##

(230) Iridium complex (0.71 mg, 0.884 mol, 0.02 equiv) was taken in dry THF (2 mL) and bubbled with nitrogen for 2 minutes at rt. The red colored catalyst dissolved in THF. Then, the solution was purged with hydrogen for about 2 more minutes. The reaction mixture color changed from red to colorless and the solution was let stir for 15 minutes under hydrogen pressure. The solution of the active catalyst was added to the of solution of allyl quinovopyranoside 79* (30 mg, 0.044 mmol, 1 equiv) in dry THF (1 mL) under argon via syringe and let stir for 2 hours at rt. The reaction mixture was quenched with NaHCO.sub.3 solution and extracted with DCM. Combined organic layers were washed with brine, dried over Na.sub.2SO.sub.4, filtered and evaporated to get the isomerized compound (isomerization confirmed by .sup.1H NMR). The substrate was taken in THF-water (2:1, 1.5 mL) and iodine (22 mg, 0.088 mmol, 2 equiv) was added at rt. The brown colored solution was stirred for 2 hours before quenched with 10% solution of Na.sub.2S.sub.2O.sub.3 solution. Then, the aqueous phase was extracted with ethyl acetate (3). Combined organic layers were dried over Na.sub.2SO.sub.4, filtered and the solvent evaporated to get brown liquid. Column chromatography on silica gel (hexanes/ethyl acetate) afforded the quinovopyranoside 80* (30 mg, 0.053 mmol, quant.). [].sub.D.sup.20=8.4 (c=1.7, CHCl.sub.3); IR .sub.max (film) 3429, 3032, 2937, 2868, 2110, 1698, 1455, 1424, 1093, 698 cm.sup.1; .sup.1H NMR (600 MHz, CDCl.sub.3) 7.87-7.80 (m, 3H), 7.79 (s, 1H), 7.51-7.45 (m, 3H), 7.42-7.13 (m, 10H), 5.18 (d, J=18.8 Hz, 2H), 4.94 (ABq, J=11.4 Hz, 2H), 4.50 (d, J=12.8 Hz, 2H), 4.31-4.18 (m, 1H), 3.92-3.78 (m, 1H), 3.54-3.34 (m, 3H), 3.34-3.12 (m, 4H), 2.49 (d, J=2.8 Hz, 1H), 1.60 (q, J=13.9, 7.5 Hz, 4H), 1.43-1.27 (m, 2H), 1.36 (d, J=6.2 Hz, 3H). .sup.13C NMR (151 MHz, CDCl.sub.3) 156.9, 156.3, 138.1, 135.5, 133.4, 133.2, 128.7, 128.6, 128.1, 128.03, 127.96, 127.9, 127.3, 127.0, 126.4, 126.2, 126.0, 101.9, 83.2, 75.3, 71.4, 70.0, 67.3, 66.7, 50.7, 50.3, 47.2, 46.3, 29.4, 28.0, 27.6, 23.3, 18.1. HRMS (ESI+) Calcd for C.sub.37H.sub.42N.sub.4O.sub.6Na.sup.+ [M+Na].sup.+ 661.3002, found 661.3019.

Example 73: Synthesis of N-(Benzyl)benzyloxycarbonyl-5-amino-pentanyl [2,3,4,6-tetra-O-benzyl--D-glucopyranosyl-(14)]-benzyl-3,4-di-O-benzyl-6-O-benzoyl--D-glucopyranosyluronate-(13)-2-azido-2-deoxy--L-fucopyranosyl-(13)-2-azido-2-deoxy-4-O-(2-naphthalenylmethyl)--D-quinovopyranoside (82*)

(231) ##STR00135##

(232) Acceptor 80* (17 mg, 0.027 mmol, 1.9 equiv) and trisaccharide imidate 68* (20 mg, 0.014 mmol, 1.0 equiv) were coevaporated with toluene two times and dried in vacuo. The residue was dissolved in DCM (1 mL). Molecular sieves acid washed (4 ) were added and the reaction mixture cooled to 35 C. TMSOTf (1 L, 5.5 mol, 0.4 equiv) was added and the reaction let warm to 20 C. over 1 h. The reaction was quenched with triethylamine, filtered and the solvent evaporated. Column chromatography on silica gel (hexanes/ethyl acetate) afforded tetrasaccharide (81*) (19.3 mg, 10.3 mol, 73%). HRMS (ESI+) Calcd for C.sub.109H.sub.117N.sub.7O.sub.22Na.sup.+ [M+Na].sup.+ 1899.8183, found 1899.8203. To a solution of tetrasaccharide (81*) (17 mg, 0.009 mmol, 1 equiv) in DCM (1 mL), a solution of hydrazine hydrate (6 L, 0.12 mmol, 13 equiv) dissolved in AcOH (0.04 mL) and pyridine (0.06 mL) was added. The resulting reaction mixture was stirred at rt for 1 to 2 h. The reaction was quenched by the addition of acetone and the solvent evaporated. Column chromatography on silica gel (hexanes/ethyl acetate) afforded tetrasaccharide 82* (14 mg, 7.87 mol, 87%). .sup.1H NMR (400 MHz, CDCl.sub.3) 7.72 (m, 3H), 7.66 (bs, 1H), 7.41-7.35 (m, 2H), 7.31-7.12 (m, 41H), 7.09-7.04 (m, 3H), 6.98-6.90 (m, 2H), 5.59 (d, J=3.7 Hz, 1H), 5.15-5.04 (m, 4H), 4.97-4.87 (m, 2H), 4.82-4.57 (m, 8H), 4.47-4.40 (m, 5H), 4.35-4.28 (m, 3H), 4.27-4.18 (m, 1H), 4.13 (d, J=7.1 Hz, 1H), 3.99-3.80 (m, 5H), 3.68-3.31 (m, 16H), 3.25-3.10 (m, 4H), 1.65-1.40 (m, 4H), 1.37-1.22 (m, 2H), 1.31 (d, J=6.1 Hz, 3H), 0.75 (d, J=6.6 Hz, 3H). HRMS (ESI+) Calcd for C.sub.104H.sub.111N.sub.7O.sub.20Na.sup.+ [M+Na].sup.+ 1801.7815, found 1801.7781.

Example 74: Synthesis of N-(Benzyl)benzyloxycarbonyl-5-amino-pentanyl 2-azido-3,4-di-O-benzyl-2-deoxy--L-pneumopyranosyl-(12)-[2,3,4,6-tetra-O-benzyl--D-glucopyranosyl-(14)]-benzyl-3,4-di-O-benzyl-6-O-benzoyl--D-glucopyranosyluronate-(13)-2-azido-2-deoxy--L-fucopyranosyl-(13)-2-azido-2-deoxy-4-O-(2-naphthalenylmethyl)--D-quinovopyranoside (83*)

(233) ##STR00136##

(234) Acceptor 82* (13.5 mg, 7.6 mol, 1.0 equiv) and imidate 8a* (10 mg, 0.019 mmol, 2.4 equiv) were coevaporated with toluene two times and dried in vacuo. The residue was dissolved in toluene (1 mL). The reaction mixture cooled to 35 C. TMSOTf (0.25 L, 1.4 mol, 0.1 equiv) was added and the reaction let warm to 20 C. over 2 h. The reaction was quenched with triethylamine, filtered and the solvent evaporated. Column chromatography on silica gel (hexanes/ethyl acetate) afforded pentasaccharide 83* (13.5 mg, 6.34 mol, 84%). HRMS (ESI+) Calcd for C.sub.124H.sub.132N.sub.10O.sub.23Na.sup.+ [M+Na].sup.+ 2152.9398, found 2152.9406.

Example 75: Synthesis of 5-amino-pentanyl 2-N-acetyl--L-pneumopyranosyl-(12)-[-D-glucopyranosyl-(14)]--D-glucopyranosyluronate-(13)-2-N-acetyl--L-fucopyranosyl-(13)-2-N-acetyl--D-quinovopyranoside (85*)

(235) ##STR00137##

(236) To a solution of azido pentasaccharide 83* (13 mg, 6.1 mol, 1 equiv) in pyridine (1 mL), thioacetic acid (0.25 mL) was added and stirred for 5 days at rt. The reaction mixture was concentrated and purified by column chromatography on silica gel (hexanes/acetone) to afford the acetamide pentasaccharide (84*) (9.4 mg, 4.31 mol, 71%). HRMS (ESI+) Calcd for C.sub.130H.sub.144N.sub.4O.sub.26Na.sup.+ [M+Na].sup.+ 2201.0000, found 2201.0027. The pentasaccharide (84*) (5.5 mg, 2.52 mol, 1 equiv) was dissolved in CH.sub.2Cl.sub.2/tBuOH/water (1.5:16:8, 1 mL), purged with argon and treated with a suspension of Pd(OH).sub.2 on carbon (20% (w/w) loading, 10 mg) in the same solvent mixture (1 mL). The suspension was purged with hydrogen and stirred under hydrogen atmosphere at rt. After 24 h, a freshly prepared suspension of Pd(OH).sub.2 (5 mg, 1 mL) was added and the reaction was stirred for 24 h more, filtered and concentrated. The residue was purified by solid phase extraction (Chromabond C18, Macherey-Nagel) and size exclusion chromatography on Sephadex LH-20 (MeOH:H.sub.2O) and lyophilized to give pentasaccharide 85* (1.5 mg, 1.49 mol, 59%) as a white solid. .sup.1H NMR (400 MHz, D.sub.2O) 5.20 (d, J=4.0 Hz, 1H), 5.09 (s, 1H), 5.01 (d, J=7.6 Hz, 1H), 4.52 (d, J=7.9 Hz, 1H), 4.51-4.29 (m, 5H), 4.18-4.13 (m, 1H), 4.13-4.07 (m, 1H), 4.02-3.90 (m, 2H), 3.86-3.62 (m, 6H), 3.58 (t, J=9.4 Hz, 1H), 3.54-3.30 (m, 9H), 3.25 (t, J=9.2 Hz, 1H), 3.13 (dd, J=9.3, 7.9 Hz, 1H), 2.97-2.92 (m, 2H), 2.01 (s, 3H), 1.97 (s, 3H), 1.96 (s, 3H), 1.68-1.49 (m, 4H), 1.40-1.31 (m, 2H), 1.31-1.23 (m, 6H), 1.07 (d, J=6.6 Hz, 3H). .sup.13C NMR (151 MHz, D.sub.2O) 175.8, 173.9, 173.6, 173.2, 102.6, 101.3, 99.5, 96.0, 93.6, 77.6, 77.1, 76.3, 75.8, 75.7, 75.3, 73.7, 73.4, 71.8, 71.5, 71.1, 70.7, 69.9, 69.7, 66.9, 66.8, 66.6, 63.7, 60.5, 55.5, 50.6, 48.7, 39.2, 28.0, 26.6, 22.2, 22.1, 22.0, 21.8, 16.6, 15.6, 15.2. HRMS (ESI+) Calcd for C.sub.141H.sub.70N.sub.4O.sub.24Na.sup.+ [M+Na].sup.+ 1025. 4278, found 1025.4309.

Example 76: 5-amino-pentanyl [-D-glucopyranosyl-(14)]--D-glucopyranosyluronate-(13)-2-N-acetyl--L-fucopyranoside (Ref. 1)

(237) ##STR00138##

(238) .sup.1H NMR (400 MHz, D.sub.2O) 4.93 (d, J=3.5 Hz, 1H), 4.67-4.56 (m, 2H), 4.30-4.27 (m, 1H), 4.26-4.03 (m, 3H), 3.92-3.84 (m, 1H), 3.76-3.60 (m, 3H), 3.54-3.25 (m, 8H), 2.94 (t, J=7.6 Hz, 2H), 1.97 (s, 3H), 1.68-1.52 (m, J=20.7, 7.0, 6.4 Hz, 4H), 1.44-1.34 (m, J=7.6 Hz, 1H), 1.31 (d, J=6.7 Hz, 3H), 1.27-1.18 (m, 1H). HRMS (ESI+) Calcd for C.sub.25H.sub.44N.sub.2O.sub.6Na.sup.+ [M+Na].sup.+ 651.2589, found 651.2598.

Example 77: 5-amino-pentanyl [-D-glucopyranosyl-(14)]--D-glucopyranosyluronate-(13)-2-N-acetyl--L-fucopyranosyl-(13)-2-N-acetyl--D-fucopyranoside (Ref. 2)

(239) ##STR00139##

(240) .sup.1H NMR (400 MHz, D.sub.2O) 5.04 (d, J=3.9 Hz, 1H), 4.65-4.60 (m, 2H), 4.38 (d, J=8.5 Hz, 1H), 4.34-4.06 (m, 4H), 3.95-3.24 (m, 16H), 2.98-2.90 (m, 2H), 1.98 (s, 3H), 1.96 (s, 3H), 1.68-1.49 (m, 4H), 1.41-1.17 (m, 2H), 1.31 (d, J=6.8 Hz, 3H), 1.23 (d, J=6.5 Hz, 3H). HRMS (ESI+) Calcd for C.sub.33H.sub.57N.sub.3O.sub.20Na.sup.+ [M+Na].sup.+ 833. 3433, found 833.3441.

Example 78: Glycan Microarray Preparation and ScreeningI

(241) 64 well glycan arrays were printed using a Scienion S3 microarray printer on CodeLink NHS activated glass slides (Surmodics). All compounds (proteins and glycans containing an aminolinker) were dissolved in printing buffer (50 mM sodium phosphate, pH 8.5). The printed slides were stored overnight in a humidity saturated chamber for complete reaction, dried by centrifugation at 1200 g and stored at 4 C. afterwards. Prior to use, slides were washed with water, quenched with quenching solution (50 mM sodium phosphate, 100 mM ethanolamine, pH 7.4) at room temperature for 2 h, washed with water and blocked for 1 h at room temperature with 1% BSA in PBS. After washing with PBS, the slides were dried by centrifugation. A 64 well gasket was applied to the slides and primary antibody incubation (serum dilutions in 1% BSA-PBS) was carried out at room temperature for 1 h. The wells were washed three times for 10 min with PBS containing 0.1% Tween-20. Secondary antibody dilutions were pipetted into the wells. After 1 h incubation at room temperature in the dark, the slides were washed three times for 10 min with PBS containing 0.1% Tween-20 and once shortly with water. The gasket was removed and the slides were dried by centrifugation. Fluorescence was read out using a GenePix 4300A microarray scanner (Bucher Biotec, Basel, Switzerland) with a 488 nm laser for FITC excitation and a 594 nm laser for AlexaFluor 594. GenePix Pro 7 (Bucher Biotec) was used for analysis.

(242) A glycan microarray prepared as above described containing eleven SP5 CPS substructures was used to identify the binding epitopes of commercial anti-SP5 specific typing sera generated in rabbits (see FIG. 4).

(243) As shown by FIG. 4A, only three out of eleven glycans were bound by specific anti-SP5 IgG, namely tetrasaccharide 33* (position 1), -PneNAc-(1.fwdarw.2)-GlcA disaccharide 27* (position 8) and -PneNAc monosaccharide 36* (position 10). All three recognized oligosaccharides contain the -PneNAc residue, demonstrating that this motif is part of the recognized epitope. It was further observed that IgG bound tetrasaccharide 33* and disaccharide 27* in comparable levels, suggesting that -PneNAc-(1.fwdarw.2)-GlcA is in fact the epitope recognized by IgG of specific SP5 typing sera. FIG. 4B shows a microarray sample where the typing serum was pre-incubated with a solution of the natural SP5 CPS. The anti-glycan IgG levels are significantly reduced compared to the sample of FIG. 4A. This means that antibodies bound to the natural CPS during pre-incubation are no longer available for binding to the synthetic glycans. Consequently, the binding epitopes of natural CPS and the synthetic glycans are identical.

(244) In order to identify epitopes recognized by the human immune system, further screenings were carried out using pooled sera of humans who had received a pneumococcal CPS-based vaccine, containing the CPS of multiple different serotypes (see FIG. 5).

(245) The IgG response of immunized humans towards the synthetic glycans is shown in FIG. 5A. Although the sera of immunized humans were not SP5 specific, they contained IgG predominantly against tetrasaccharide 33* (position 1). IgG binding to PneNAc-(1.fwdarw.2)-GlcA disaccharide 27* (position 8) and PneNAc 36* (position 10) were also observed, however at lower levels.

(246) Additionally, low levels of anti-21* (position 5) and anti-51* (position 6) IgG and very low levels of anti-20* (position 4), anti-44* (position 7), anti-52* (position 9) and anti-37* (position 11) IgG were detected. Antibodies against D-FucNAc 15* (position 3) and the Sug-containing sample 14* (position 2) were not detected at all. Interestingly, higher levels of anti-disaccharide 21* antibodies were detected than for the sample containing L-FucNAc-(1.fwdarw.3)--Sug 20*. This would suggest that the presence of the ketone is indeed detrimental to antibody binding. Pre-incubation of the human sera with the natural SP5 CPS, shown in FIG. 5B, led to a significant reduction of anti-tetrasaccharide 33* IgG levels. This proves that the antibodies binding 33* are SP5 specific since they also bind the SP5 CPS.

(247) In conclusion, the microarray analyses revealed that the terminal PneNAc residue is part of the epitope recognized by anti-SP5 CPS antibodies and that -PneNAc-(1.fwdarw.2)-GlcA is the predominant epitope recognized by SP5 typing sera. In the case of vaccinated humans, the SP5-specific epitope seems to be larger, resembling tetrasaccharide 33*. The reducing end D-FucNAc or Sug residues do not seem to be important for the immunological response to SP5.

Example 79: Conjugation of Tetrasaccharide 33* to CRM197: Synthesis of Conjugate 58*a, 58*b and 58*c

(248) Conjugates were prepared using a two-step procedure as described here in details.

Step 1: Formation of the p-Nitro Phenyl (PNP) Amide

(249) To the tetrasaccharide 33* (0.5 mg, 0.6 mol, 1 equivalent) and diphenyl adipate (1.6 mg, 4.2 mol, 7 equivalents) in a glass vial were added a mixture of pyridine and DMSO (1:1, 0.24 mL) and the mixture let stir for 5 minutes for complete solubilization. Then, triethylamine (0.83 L, 6 mol, 10 equivalents) was added and let stir for 20 minutes. TLC indicated complete consumption of the starting material. The solvent was removed in vacuum. The residue was washed with dichloromethane (31 mL) to remove PNP ester excess and the white solid obtained was dried in vacuum.

Step 2: Conjugation with CRM197

(250) The compound obtained at step 1 (40 equivalents) was dissolved in DMSO (10 L), CRM.sub.197 (1 equivalent) was added in sodium phosphate buffer (0.1 NaPi, pH 8.0) and the mixture let stir for 18 h. Then, the reaction mixture was washed twice with 400 L of buffer (pH 8.0), followed by washing with 400 L of autoclaved water three times using amicon filter (10000 KDa). The material was transferred to Eppendorf vial using PBS buffer and stored at 20 C. (400 L).

(251) The same procedure was repeated with buffer pH 8.5.

(252) ##STR00140##

(253) Pentasaccharide conjugate 87* was obtained by conjugation of pentasaccharide 73* to CRM.sub.197.

(254) ##STR00141##

(255) Maldi analysis: 1) Conjugate 58* (conjugate 1 in FIG. 6 B): pH 8.0, 0.9 mg CRM.sub.197, tetrasaccharide loading average=7 2) Conjugate 58*b (conjugate 2 in FIG. 6 B): pH 8.5, 0.6 mg CRM.sub.197, tetrasaccharide loading average=8 3) Conjugate 58*c (conjugate 2 in FIG. 7 A): tetrasaccharide loading average=11.2 4) Conjugate 87* (conjugate in FIG. 7 B): at pH 8.0, pentasaccharide loading average=12

(256) Characterization of Conjugates

(257) Maldi Analysis:

(258) The average molecular size of conjugates 58*a and 58*b was determined by Matrix-assisted laser desorption/ionization (MALDI) analysis using CRM.sub.197 as standard and calculate the average number of tetrasaccharide attached per CRM.sub.197 molecule.

(259) SDS-PAGE:

(260) The conjugates were resolved by SDS-PAGE (10%) in denaturing condition. The samples were prepared in 6SDS-PAGE sample loading dye. The electrophoresis was carried out at 120 V and 25 mA for 1 hr 30 min in electrode buffer and gel was stained with Coomassie brilliant blue R250.

(261) Protein Estimation

(262) The protein concentration was estimated using Micro BCA Protein Assay Kit (Thermo-scientific, USA) following the manufacturer's instructions. The sample was prepared in PBS and mixed with equal volume of reagent mixture (B:C:A::24:1:25). The plate was incubated at 37 C. and the absorbance was measured at 560 nm. The standard curve was plotted with known concentration of BSA provided with the kit.

(263) Mice Immunization and Generation of Polyclonal Sera

(264) Ten to twelve week old female ZIKA rabbits (n=3) were immunized subcutaneously with the conjugates in alum (10 g conjugate in alum (aluminum hydroxide)) formulation at day 0, 14 and 28. Preimmune and hyperimmune sera were collected at day 0, 14, 21 and 35. Control group received only CRM.sub.197 in alum formulation. The immune responses were analyzed by glycan microarray and ELISA.

Example 80: Glycan Microarray Preparation and ScreeningII

(265) Preparation of Microarrays Slides:

(266) Oligosaccharides, polysaccharides, CRM.sub.197 and spacer bearing glass slides were prepared, quenched, blocked with 1% BSA-PBS and stored at 4 C. until use.

(267) The CodeLink NHS activated glass slides (Surmodics) were spotted with synthetic glycans and native polysaccharides at two different concentrations (100 M and 200 M) in printing buffer (50 mM sodium phosphate, pH 8.5) by using a S3 piezoelectric microarray printer (Scienion) equipped with a type 4 coated nozzle. The relative humidity of spotted chamber was constantly maintained at 65%. The spotted slides were incubated over night at room temperature in a humidifying chamber. The unreactive groups on the slides were blocked with 50 mM sodium phosphate, 100 mM ethanolamine pH 9.0 at room temperature for one hour. Slides were subsequently washed three times for 5 min with water, dried by centrifugation at 300 g for 5 min (CombiSlide system, Eppendorf) and stored at 4 C. until use.

(268) Microarray Binding Assays:

(269) A FlexWell 64 (Grace Bio-Labs, Bend, Oreg., USA) grid was applied to microarray slides (The grid was applied on the printed slides as mentioned above). Slides were incubated with polyclonal sera raised in rabbits against SP-5 tetrasaccharide and pentasaccharide conjugates at different dilutions, diluted in 1% BSA-PBS (w/v) in a humid chamber for 1 h at room temperature, washed three times with 0.1% Tween-20 in PBS (v/v) and dried by centrifugation (300g, 5 min). Slides were incubated with a fluorescence-labeled secondary antibody, goat anti-mouse IgG 635 nm (red; 1 in 400 dilution), goat anti mouse IgM 594 nm (yellow; 1 in 200 dilution) and goat anti-rabbit FITC488 (1 in 200 dilutions) (Life Technologies, USA) diluted in 1% BSA in PBS (w/v) in a humid chamber for 1 h at room temperature, washed three times with 0.1% Tween-20 in PBS (v/v), rinsed once with deionized water and dried by centrifugation (300g, 5 min) prior to scanning with a GenePix 4300A microarray scanner (Molecular Devices, Sunnyvale, Calif., USA). Image analysis was carried out with the GenePix Pro 7 software (Molecular Devices). The photomultiplier tube (PMT) voltage was adjusted such that scans were free of saturation signals (FIG. 8).

(270) Further microarray was preparte to identify the smallest glycan structure of CPS-5 polysaccharide, we incubated the microarray slides with ST5 type sera raised in rabbit (FIG. 8). The reactivity pattern of type sera against printed glycan's and polysaccharide was analyzed by glycan microarray. The microarray data indicated that the type sera recognized the several synthetic glycans along with CPS-5 polysaccharide (FIG. 8B). However the type sera also recognized the tetrasaccharide 33* and pentasaccharide 73*/85* with high affinity which are considered to be the B cell epitopes that has been presented by the MHCII molecules. Thus, the following results suggest that these oligosaccharides are critical for immunity.

(271) ##STR00142## ##STR00143##

Example 81: Glycan Microarray with Antibodies Raised in Rabbit Against ST5 Synthetic Glycan Conjugates

(272) To analyze the ST5 synthetic glycans specific immune response, we subjected hyperimmune sera raised in rabbit (n=3) immunized with ST5 tetrasaccharide (58*c) and pentasaccharide conjugates (87*) to microarray slides. The microarray data suggested that the synthetic glycans specific antibodies were cross-reactive with the structures printed on slides and native polysaccharide (FIGS. 9B and D). This result suggested that the tetrasaccharide 33* and pentasaccharide 73* are immunogenic in rabbit and induced cross-reactive antibodies.

Example 82: ELISA

(273) The end point titer of rabbit sera were analyzed by ELISA. High binding ninety six well polystyrene microtiter plates (Corning, USA) were coated overnight at 4 C. with CSP-5 (50 l of 10 g/ml per well) in PBS, pH 7.2. The plates were washed thrice with PBS containing 0.1% Tween-20 (PBST) and blocked with 10% FCS in PBS at 37 C. for 1 hr. After washing thrice with PBST, the plate was incubated with the individual and pooled rabbit serum (n=3) at different dilutions in duplicate or triplicates at 37 C. for 1 hr. The plate was washed 4-5 times with PBST and incubated with HRP conjugated goat anti-rabbit IgG antibodies (diluted 1 in 10,000 in PBS containing serum diluted in 10% FCS followed by incubation at 37 C. for 1 hr. The plate was washed thoroughly with PBST and developed using 3,3,5,5-tetramethylbenzidine (Thermo Fisher Scientific, USA). The reaction was stopped by adding 2% H.sub.2SO.sub.4 and absorbance recorded at 450 nm.

(274) Antibody Responses Analyzed by ELISA

(275) Three groups of rabbit (n=3) were immunized with 10 g of glycans equivalent conjugate on day 0, 14 and 28 subcutaneously, and the antibody responses were analyzed at different time point. The CSP-5 (Life Technologies, USA) polysaccharide specific endpoint antibody titer of the individual rabbit serum was determined. Immunization with tetrasaccharide and pentasaccharide conjugates induced high antibody titers (FIG. 10A). We further quantitated the polysaccharide specific immunoglobulins by endpoint (FIG. 10B). Endpoint titer was calculated as the reciprocal of the highest serum dilution that gave an absorbance value obtained with the preimmune sera (diluted 1:1000). The antibody response for individual animal of each groups were plotted as fold change at day 35. This analysis suggested that pentasaccharide 73* or conjugate 87* is more immunogenic compared to tetrasaccharide 33* or conjugate 58*c.

Example 83 Surface Binding of Anti-ST5 Glycan Conjugates Sera

(276) The protective immunity against pneumococci is primarily antibody mediated and for an effective vaccine candidate the B cell epitopes should be accessible to the antibody. To investigate the accessibility of B cell epitope, we performed the flow cytometry for surface staining. The UV inactivated pneumococcal cells were incubated with preimmune and hyperimmune sera followed by the FITC-conjugated goat anti-rabbit antibodies. The stained bacteria were analyzed by flow cytometry. The flow cytometry data indicated that the pneumococcal cells incubated with hyperimmune sera exhibited the surface staining (tetrasaccharide; black histogram and pentasaccharide; gray histogram). We did not observe any staining with the preimmune sera (doted black histogram). This indicated that antibodies against the anti-ST5 glycans localized the epitope on the surface of pneumococci (FIG. 11) and considered to be a novel synthetic vaccine candidate.

(277) Flow Cytometry Analysis:

(278) S. pneumoniae serotype 5 cells were stained as previously described with minor modifications (Khan et al; 2015, Clin Vaccine Immunol. 22(1):99-107). Briefly, mid-logarithmic phase (A.sub.600=0.3-0.4) pneumococcal cells were harvested by centrifuging at 6000g. Cells were washed with PBS (pH 7.4) and UV inactivated for 10 min. The cell inactivation was confirmed by plating on blood agar plats. The cells were blocked with 2% BSA prepared in PBS at room temperature for 30 min followed by incubation with preimmunre and hyperimmune pooled sera (n=3) (diluted 1:100) at room temperature for 1 hr. FITC-conjugated goat anti-rabbit IgG (Life Technologies, USA) diluted 1:200 dilution in 0.5% BSA-PBS) was used as the secondary antibody. The stained cells were analyzed by flow cytometry (BD Bioscience).

Example 84: In Vitro Opsonophagocytic Killing Assay

(279) opsonophagocytic killing assay was performed as described previously (Romero-Steiner et al., CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY 1997, 415-422). Briefly, 410.sup.5 cell/ml HL-60 cells were differentiated into HL-60 cells were differentiated into phagocytic cells by using 0.8% Dimethylformamide (DMF) for 5-6 days before performing the assay. After differentiation the cells were harvested and counted by using a haemocytometer and resuspended in opsonophagocytic buffer (HBSS with Ca.sup.++ and Mg.sup.++ and 0.1% gelatin) at a density of 110.sup.7 cells/ml. Serotype 5 strain (1000 cfu) in 20 l of opsonophagocytic buffer were incubated with 10 l of pooled (n=3) anti-SP-5 tetrasaccharide conjugate (58*c) polyclonal sera (day 35). Preimmune sera (day 0), anti-CRM.sub.197 and anti-Prevnar 13 sera were used as positive and negative controls and bacteria were preopsonized for 15 min at 37 C. After preopsonization 8 l of baby rabbit complement and 40 l differentiated HL-60 cells (1:400 ratio) were added with antibody treated pneumococci for 45 min at 37 C. and 5% CO.sub.2 with intermittent shaking. The viable extracellular pneumococci were determined by plating serial dilutions on TSA plates in triplicates. The mean of 3 independent experiments was plotted as percent killing relative to the no sera control.

(280) Anti-ST5 (Tetrasaccharide) Antibodies Augment Uptake of Pneumococci by HL-60 Cells

(281) Anti-ST5 (tetrasaccharide) may facilitate clearance of pneumococci by phagocytosis. We performed opsonophagocytic killing assay to assess the functional relevance of the antibodies induced in response to immunization with ST5 (tetrasaccharide) conjugate 58*c. Differentiated HL-60 cells were incubated with serotype 5 bacteria pre-opsonized with either anti-ST5 (tetrasaccharide) or preimmune sera. The killing of pneumococci was assessed by plating method as described in the Material and Methods. The relative percent killing value obtained for polyclonal antibody against ST5 (tetrasaccharide) was greater than 50% compared to the preimmune sera (FIG. 12). We observed some basal level of killing (25%) with the preimmune sera. The anti-prevnar 13 rabbit sera were used as a positive control. The OPKA data demonstrates that ST5 (tetrasaccharide) conjugate (58*c) generate neutralizing antibodies and promote the killing of pneumococci by differentiated HL-60 cells.