Synthetic vaccines against Streptococcus pneumoniae type 1

Abstract

The present invention relates to the total synthesis of saccharide structures contained in the capsular polysaccharide of Streptococcus pneumoniae type 1, to glycoconjugates containing said saccharide structures obtained by total synthesis and to use of such glycoconjugates and pharmaceutical compositions thereof in the immunization against diseases associated with bacteria containing said saccharide structures in their capsular polysaccharide, and more specifically associated with Streptococcus pneumoniae.

Claims

1. A saccharide of general formula (I): ##STR00147## wherein A represents (CH.sub.2).sub.o1; o1 represents an integer selected from 1, 2, 3, 4, 5 and 6; M, N and P represent independently of each other one of the following sugar fragments: ##STR00148## wherein the sugar fragments S1, S2, S3 are connected to each other and to O-A-SH fragment via O-glycosidic bonds, each sugar fragment S1, S2, and S3 is not more than once present in the general formula (I), sugar fragment Si cannot be simultaneously connected to O-A-SH and sugar fragment S3, sugar fragment S3 cannot be simultaneously connected to O-A-SH and sugar fragment S2, and sugar fragment S2 cannot be simultaneously connected to O-A-SH and sugar fragment S1, and n1, n2 and n3 are integers selected from 0 and 1, wherein at least one of the integers n1, n2 and n3 is 1 and pharmaceutically acceptable salts of these saccharides.

2. A synthesis of the saccharide of the general formula (I): ##STR00149## wherein A represents (CH.sub.2).sub.o1; o1 represents an integer selected from 1, 2, 3, 4, 5 and 6; M, N and P represent independently of each other one of the following sugar fragments: ##STR00150## wherein the sugar fragments S1, S2, S3 are connected to each other and to O-A-SH fragment via O-glycosidic bonds, each sugar fragment S1, S2, and S3 is not more than once present in the general formula (I), sugar fragment Si cannot be simultaneously connected to O-A-SH and sugar fragment S3, sugar fragment S3 cannot be simultaneously connected to O-A-SH and sugar fragment S2, and sugar fragment S2cannot be simultaneously connected to O-A-SH and sugar fragment S1, and n1, n2 and n3 are integers selected from 0 and 1, wherein at least one of the integers n1, n2 and n3 is 1 and comprising : A1) Reacting the compound 2 of the formula: ##STR00151## wherein P.sup.1-P.sup.3 represent protecting groups, with the compound 3 of the formula ##STR00152## wherein P.sup.4 represents a protecting group, in order to obtain compound 4 of general formula: ##STR00153## wherein P.sup.1-P.sup.4 and A are defined as above; and performing removal of protecting groups P.sup.1-P.sup.4 on compound 4 to afford monosaccharide disulfide 5 of general formula: ##STR00154## wherein A is defined as above, and wherein monosaccharide disulfide 5 is further treated with a reducing agent to afford monosaccharide 6 of general formula: ##STR00155## wherein A is defined as above; or performing selective deprotection on compound 4 to afford compound 7 of general formula ##STR00156## wherein P.sup.5 is a protecting group and P.sup.1, P.sup.3, P.sup.4 and A are defined as above; or A2) Reacting compound 8 of general formula ##STR00157## wherein P.sup.6 and P.sup.7 represent protecting groups, with compound 3 to afford compound 9 of general formula ##STR00158## wherein P.sup.6, P.sup.7 and A are defined as above; and performing conversion of the azido group to acetamido group and removal of the protecting groups P.sup.4, P.sup.6 and P.sup.7 on compound 9 to afford monosaccharide disulfide 10 of general formula: ##STR00159## wherein A is defined as above, and wherein monosaccharide disulfide 10 is further treated with a reducing agent to afford monosaccharide 11 of general formula: ##STR00160## wherein A is defined as above; or performing selective deprotection on compound 9 to afford compound 12 of general formula: ##STR00161## wherein P.sup.4, P.sup.7 and A are defined as above; or A3) Reacting compound 13 of general formula ##STR00162## wherein P.sup.8-P.sup.11 represent protecting groups, with compound 3 to afford compound 14 of general formula: ##STR00163## wherein P.sup.4, P.sup.8-P.sup.11 are defined as above; and performing selective deprotection of compound 14 to afford compound 15 of general formula: ##STR00164## wherein P.sup.4, P.sup.8, P.sup.9, P.sup.11 and A are defined as above; and B1) Reacting compound 7 with compound 13 to afford compound 16 of general formula: ##STR00165## wherein P.sup.1, P.sup.3-P.sup.5, P.sup.8-P.sup.11 and A are defined as above; and performing removal of protecting groups P.sup.1 P.sup.3-P.sup.5, P.sup.8-P.sup.11 on compound 16 to afford disaccharide disulfide 17 of general formula: ##STR00166## wherein A is defined as above and wherein disaccharide disulfide 17 is further treated with a reducing agent to afford disaccharide 18 of general formula: ##STR00167## wherein A is defined as above; or performing selective removal of protecting group P.sup.10 on compound 16 to afford compound 19of general formula: ##STR00168## wherein P.sup.1, P.sup.3-P.sup.5, P.sup.8, P.sup.9, P.sup.11 and A are defined as above; or B2) Reacting compound 15 with compound 8 to afford compound 20 of general formula: ##STR00169## wherein P.sup.4, P.sup.6-P.sup.9, P.sup.11 and A are defined as above; and performing conversion of the azido group to acetamido group and removal of the protecting groups P.sup.4, P.sup.6-P.sup.9, P.sup.11 on compound 20 to afford disaccharide disulfide 21 of general formula: ##STR00170## wherein A is defined as above and wherein disaccharide disulfide 21 is treated with a reducing agent to afford disaccharide 22 of general formula: ##STR00171## wherein A is defined as above; or performing selective removal of protecting group P.sup.6 on compound 20 to afford compound 23of general formula: ##STR00172## wherein P.sup.4, P.sup.7-P.sup.9, P.sup.11 and A are defined as above; or B3) Reacting compound 12 with compound 2 to afford compound 24 of general formula: ##STR00173## wherein P.sup.1-P.sup.4, P.sup.7 and A are defined as above, and performing conversion of the azido group to acetamido group and removal of protecting groups P.sup.1-P.sup.4 and P.sup.7 on compound 24 to afford disaccharide disulfide 25 of general formula: ##STR00174## wherein A is defined as above, and wherein disaccharide disulfide 25 is further treated with a reducing agent to afford disaccharide 26 of general formula: ##STR00175## wherein A is defined as above; or performing selective deprotection on compound 24 to afford compound 27 of general formula: ##STR00176## wherein P.sup.12 is a protecting group and P.sup.1, P.sup.3, P.sup.4, P.sup.7 and A are defined as above; and C1) Reacting compound 19 with compound 8 to afford compound 28 of general formula: ##STR00177## wherein P.sup.1, P.sup.3-P.sup.9, P.sup.11 and A are defined as above; and wherein protecting group P.sup.6 is replaced with protecting group P.sup.13 in order to obtain compound 29 of the following chemical formula: ##STR00178## wherein P.sup.1, P.sup.3-P.sup.5, P.sup.7-P.sup.9, P.sup.11, P.sup.13 and A are defined as above; and conversion of compound 29 to trisaccharide disulfide 30 by conversion of the azido group in the acetamido group and cleavage of the protecting group P.sup.1, P.sup.3-P.sup.5, P.sup.7-P.sup.9, P.sup.11, .sup.13, wherein compound 30 is of general formula: ##STR00179## and wherein A is defined as above; and conversion of trisaccharide disulfide 30 to trisaccharide 31 by treatment with a reducing agent, wherein compound 31 is of general formula: ##STR00180## and wherein A is defined as above; or C2) Reacting compound 23 with compound 2 to afford compound 32 of general formula: ##STR00181## wherein P.sup.1-P.sup.4, P.sup.7-P.sup.9, P.sup.11 and A are defined as above; and conversion of compound 32 to trisaccharide disulfide 33 by conversion of the azido group in the acetamido group and cleavage of the protecting group P.sup.1-P.sup.4, P.sup.7-P.sup.9, P.sup.11, wherein compound 33 is of general formula: ##STR00182## wherein A is defined as above; and conversion of trisaccharide disulfide 33 to trisaccharide 34 by treatment with a reducing agent, wherein compound 34 is of general formula: ##STR00183## wherein A is defined as above; or C3) Reacting compound 27 with compound 13 to afford compound 35 of general formula: ##STR00184## wherein P.sup.1, P.sup.3, P.sup.4, P.sup.7-P.sup.11 and A are defined as above; and conversion of compound 35 to trisaccharide disulfide 36 by conversion of the azido group in the acetamido group and cleavage of the protecting group P.sup.1, P.sup.3, P.sup.4, P.sup.7-P.sup.11 wherein compound 36 is of general formula: ##STR00185## wherein A is defined as above; and conversion of trisaccharide disulfide 36 to trisaccharide 37 by treatment with a reducing agent, wherein compound 37 is of general formula: ##STR00186## wherein A is defined as above.

3. The synthesis according to claim 2 further comprising: preparing a salt of the compound of general formula (I) or preparing a lyophilisate of the compound of general formula (I) or of the salt of the compound of general formula (I).

4. The synthesis according to claim 2, wherein the reactions between compounds 2 and 3, compounds 2 and 12, and compounds 2 and 23 are performed in presence of DMTST and TTBPy in a mixture of non-polar and polar aprotic solvents.

5. The synthesis according to claim 2, wherein the replacement of protecting group P.sup.6 on compound 28 with protecting group P.sup.13 to obtain compound 29 is performed in two steps, first involving the reaction of compound 28 with hydrazine or a hydrazinium salt in a solvent or a mixture of solvents, and second by treatment of the product obtained after the first step with BnOCH.sub.2SCy, DMTST and TTBPy in a non-polar solvent.

6. The synthesis according to claim 2, wherein the cleavage of the protecting groups involves first cleavage of the base-labile protecting groups by treatment with a base in a mixture of polar aprotic and polar protic solvents; and second cleavage of the protecting groups sensitive to hydrogenation by exposure to sodium and ammonia in a mixture of polar protic and polar aprotic solvents.

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

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1 shows the global distribution of Streptococcus pneumoniae serotypes

(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 printing pattern of the saccharides of general formula (I) on microarray slides

(5) FIG. 5 shows the binding of human pneumococcal serum 007sp (pooled sera of 287 humans immunized with Pneumovax vaccine) to saccharides of general formula (I), which are coated on modified CodeLink NHS slides in presence and in absence of native Streptococcus pneumoniae type 1 polysaccharide.

(6) FIG. 6 shows the binding of the rabbit anti-Streptococcus pneumoniae type 1 typing serum to saccharides of general formula (I), which are coated on modified amine-coated GAPS II slides (Corning) in presence and in absence of native Streptococcus pneumoniae type 1 polysaccharide.

(7) FIG. 7 shows the binding of the rabbit anti-Streptococcus pneumoniae type 1 typing serum to saccharides of general formula (I), which are coated on modified amine-coated GAPS II slides (Corning).

(8) FIG. 8 shows the binding of the rabbit anti-Streptococcus pneumoniae type 1 typing serum to saccharides of general formula (I), which are coated on modified CodeLink NHS slides.

(9) 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.

(10) 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

(11) Chemical Synthesis

(12) General information for chemical synthesis. 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 glass 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.

Example 1

4-(Benzyloxycarbonyl)amino-3-O-levulinoyl-4,6-dideoxy-D-galactal (1*)

(13) ##STR00102##

(14) To a stirred solution of 4-O-(benzyloxycarbonyl)amino-3-hydroxy-4,6-dideoxy-D-galactal (Org. Lett. 2010, 12, 1624) (1.64 g, 6.21 mmol) (1.64 g, 6.21 mmol) in CH.sub.2Cl.sub.2 (40 ml) were added at 0 C. pyridine (0.501 ml, 6.21 mmol), levulinic acid (0.96 ml, 9.31 mmol), DMAP (0.152 g, 1.242 mmol) and EDC (1.205 ml, 6.83 mmol). The mixture was warmed to room temperature and stirred at that temperature. After 3 h, 0.5 eq. levulinic acid and 0.5 eq. EDC were added to drive the reaction to completion. After 5 h, the mixture was diluted with 100 ml DCM and washed with water (50 ml), sat. aq. NH.sub.4Cl (50 ml), sat. aq. NaHCO.sub.3 (50 ml) and brine (50 ml). The organic fraction was dried over Na.sub.2SO.sub.4 and concentrated. The residue was purified by flash chromatography (EtOAc/hexanes 1:1) to give ester 1* (2.07 g, 5.73 mmol, 92%) as a clear oil. HRMS (ESI) calcd for C.sub.19H.sub.23NO.sub.6 (M+Na+) 384.1423. found 384.1415 m/z.

Example 2

Dibutyl [2-azido-4-(benzyloxycarbonyl)amino-3-O-levulinoyl-2,4,6-trideoxy-D-galactopyranosyl] phosphate (2*)

(15) ##STR00103##

(16) To a stirred solution of galactal 1* (3.17 g, 8.77 mmol) in dry MeCN (44 ml) were added at 25 C. ceric ammonium nitrate (14.42 g, 26.3 mmol) and sodium azide (0.86 g, 13.15 mmol). The reaction was stirred vigorously between 25 C. and 20 C. for 6 h. The mixture was diluted with cold Et.sub.2O (50 ml). The organic layer was washed with cold water (330 ml), dried over Na.sub.2SO.sub.4 and concentrated. The residue was filtered through a plug of silica gel (EtOAc/hexanes/Et.sub.3N 1:1:0.01) to give the crude glycosyl nitrate as 4:1 galactoltalo mixture (2.01 g) as a slightly yellow oil.

(17) To the crude glycosyl nitrate (2.01 g) was added at room temperature a solution of cesium dibutyl phosphate (2.21 g, 6.45 mmol) in dry DMF (28 ml). The mixture was stirred at that temperature for 4.5 h, diluted with EtOAc (100 ml) and poured into water (100 ml). The organic phase was washed with water (550 ml) and the combined aqueous fractions were extracted with EtOAc (50 ml). The organic phase was dried over Na.sub.2SO.sub.4 and concentrated. The residue was purified by flash chromatography (EtOAc/hexanes 45:55 to 50:50) to give glycosyl phosphate 2* (1.84 g, 3.00 mmol, 37%, 1:10 /) as a clear oil. HRMS (ESI) calcd for C.sub.27H.sub.41N.sub.4O.sub.10P (M+Na.sup.+) 635.2458. found 635.2422 m/z.

Example 3

Ethyl 2-O-benzyl-3,4-isopropylidene-1-thio--D-galactopyranoside (3*)

(18) ##STR00104##

(19) To a stirred solution of ethyl 6-O-tert-butyldimethylsilyl-3,4-isopropylidene-1-thio--D-galactopyranoside (Bioorg. Med. Chem. 2001, 9, 1395) (45.7 g, 121 mmol) in DMF (150 ml) and THF (75 ml) were added at 0 C. portionwise sodium hydride (60%, 7.24 g, 181 mmol) and then benzyl bromide (17.2 ml, 145 mmol). The mixture was stirred for 1 h at 0 C., slowly warmed to room temperature and stirred for 16 h at that temperature. The reaction was quenched at 0 C. with sat. aq. NH.sub.4Cl (20 ml), diluted with water (200 ml) and EtOAc (150 ml) and stirred for 15 min at 0 C. After separation, the organic phase was washed with water (5100 ml) and the combined aqueous fractions were re-extracted with EtOAc (2100 ml). The combined organic extracts were dried over Na.sub.2SO.sub.4 and concentrated to give the crude benzyl ether (61 g) as a yellow oil.

(20) To a stirred solution of the crude benzyl ether (61 g) in THF (370 ml) was added at 0 C. tetrabutylammonium fluoride (1 M in THF, 166 ml, 166 mmol). The mixture was warmed to room temperature and stirred for 1 h. The reaction was diluted with sat. aq. NaHCO.sub.3 (200 ml) and EtOAc (100 ml). After separation, the aqueous phase was extracted with EtOAc (3100 ml), the combined organic fractions were dried over MgSO.sub.4 and concentrated. The residue was purified by flash chromatography (EtOAc/hexanes 0:1 to 1:3 to 1:1) to give alcohol 3* as a white solid. HRMS (ESI) calcd for C.sub.18H.sub.26O.sub.5S (M+Na.sup.+) 377.1398. found 377.1416 m/z.

Example 4

Methyl (ethyl 2-O-benzyl-1-thio--D-galactopyranosid)uronate (4*)

(21) ##STR00105##

(22) To a vigorously stirred solution of alcohol 4* (6.0 g, 16.93 mmol) in CH.sub.2Cl.sub.2 (50 ml) and H.sub.2O (25 ml) were added at 0 C. TEMPO (0.53 g, 3.39 mmol) and BAIB (10.9 g, 33.9 mmol). The mixture was warmed to room temperature and stirred for 1 h at that temperature. The reaction was quenched with 10% aq. Na.sub.2S.sub.2O.sub.3 (10 ml) and diluted with EtOAc (30 ml). After separation, the organic phase was washed with 10% Na.sub.2S.sub.2O.sub.3 (420 ml). The aqueous phase was extracted with EtOAc (220 ml) and the combined organic fractions were dried over Na.sub.2SO.sub.4 and concentrated to give the crude acid (7.92 g) as yellow oil.

(23) To a stirred solution of acetyl chloride (6.04 ml, 85 mmol) in MeOH (300 ml) was added dropwise at 0 C. a solution of the crude acid (7.92 g) in MeOH (40 ml). The mixture was warmed to room temperature, stirred for 2 h at that temperature and cooled to 0 C. The reaction was quenched with sat. aq. NaHCO3 (30 ml) and neutralized to pH 7 with solid NaHCO.sub.3. The volatiles were evaporated and the mixture was diluted with EtOAc (70 ml). After separation, the aqueous phase was extracted with EtOAc (550 ml). The combined organic fractions were dried over Na.sub.2SO.sub.4 and concentrated. Flash chromatography was performed (EtOAc/hexanes 2:3 to 1:1, then 1:0) to give the crude product, which was crystallized in methanol at 20 C. (5 ml/g crude product) to give diol 4* (3.47 g, 10.13 mmol, 60%) as a white solid. HRMS (ESI) calcd for C.sub.16H.sub.22O.sub.6S (M+Na).sup.+ 365.1034. found 365.1058 m/z.

Example 5

Methyl (ethyl 2-O-benzyl-3,4-O-endo-benzylidene-1-thio--D-galactopyranosid)urinate (5*) and Methyl (ethyl 2-O-benzyl-3,4-O-exo-benzylidene-1-thio--D-galactopyranosid)uronate (6*)

(24) ##STR00106##

(25) To a stirred solution of diol 4* (2.99 g, 8.73 mmol) in dry acetonitrile (29 ml) were added at room temperature benzaldehyde dimethyl acetal (6.57 ml, 43.6 mmol) and DL-camphorsulfonic acid (0.51 g, 2.18 mmol). The mixture was stirred at room temperature for 5 h and the reaction was quenched by addition of triethylamine (0.35 ml). The mixture was concentrated under reduced pressure to give a residue, which was filtered through a short plug of silica gel (hexanes/EtOAc 8:1 (2% Et.sub.3N) to 1:1 (2% Et.sub.3N)) to give a 1:1 mixture of benzylidene acetals 5* (endo) and 6* (exo) (3.46 g, 8.03 mmol, 92%). The isomers were separated by selective crystallization of exo-isomer 6* from EtOAc/hexanes and chromatographic separation of the mother liquor (Biotage, flat gradient of 10% to 40% EtOAc in hexanes+0.5% Et.sub.3N). Analytical data for 5*: Clear oil. HRMS (ESI) calcd for C.sub.23H.sub.26O.sub.6S (M+Na).sup.+ 453.1348. found 453.1352 m/z. Analytical data for 6*: White solid. HRMS (ESI) calcd for C.sub.23H.sub.26O.sub.6S (M+Na).sup. 453.1348. found 453.1338 m/z.

Example 6

Methyl (ethyl 2,3-O-benzyl-1-thio--D-galactopyranosyl)uronate (7*)

(26) ##STR00107##

(27) To a solution of acetal 6* (162 mg, 0.38 mmol) and sodium cyanoborohydride (296 mg, 4.70 mmol) in THF (9.4 ml) was added at room temperature a solution of hydrogen chloride (1 M in Et.sub.2O) until the evolution of gas ceased. After 10 min, sodium cyanoborohydride (296 mg, 4.70 mmol) was added, followed by the addition of HCl. The reaction was stirred at room temperature, diluted with EtOAc (30 ml) and quenched with sat. aq. NaHCO.sub.3 (30 ml). After separation, the organic layer was washed with sat. aq. NaHCO.sub.3 (20 ml) and the aqueous layer was re-extracted with EtOAc (220 ml). The organic extracts were pooled, dried over MgSO.sub.4 and concentrated. The residue was purified by flash chromatography (EtOAc/hexanes 1:1) to give alcohol 7* (67.5 mg, 0.156 mmol, 42%) as a white solid. HRMS (ESI) calcd for C.sub.23H.sub.28O.sub.6S (M+Na.sup.+) 455.1504. found 455.1511 m/z.

Example 7

Methyl (ethyl 2,3-O-benzyl-4-O-fluorenylmethoxycarbonyl-1-thio--D-galactopyranosyl uronate (8*)

(28) ##STR00108##

(29) To a stirring solution of alcohol 7* (160 mg, 0.370 mmol) in pyridine (1.2 ml) was added at 0 C. FmocCl (383 mg, 1.48 mmol). The mixture was warmed to room temperature and stirred for 3 h. The mixture was then diluted with EtOAc (50 ml) and washed with 1 N HCl (230 ml) and sat. aq. NaHCO.sub.3 (30 ml). The organic phase was dried over Na.sub.2SO.sub.4 and concentrated. The residue was purified by flash chromatography (EtOAc/hexanes 1:2) to give carbonate 8* (217 mg, 0.331 mmol, 90%) as a white foam. HRMS (ESI) calcd for C.sub.38H.sub.38O.sub.8S (M+Na).sup.+ 677.2185. found 677.2167 m/z.

Example 8

Dibutyl [methyl (2,3-O-benzyl-4-O-fluorenylmethoxycarbonyl-/-D-galactopyranosyl)uronate] phosphate (9*)

(30) ##STR00109##

(31) Thioglycoside 8* (200 mg, 0.305 mmol) was co-evaporated with dry toluene (230 ml), kept under high vacuum for 1 h and dissolved in dry CH.sub.2Cl.sub.2 (3 ml). Activated molecular sieves (3 -AW) were added and the solution was stirred for 15 min at room temperature. The solution was then cooled to 0 C., treated with dibutyl phosphoric acid (128 mg, 0.611 mmol) and stirred for another 15 min. The mixture was then treated with NIS (89 mg, 0.397 mmol), warmed to room temperature and stirred for 3 h at that temperature. The reaction was diluted with CH.sub.2Cl.sub.2 (20 ml) and quenched with a 1:1 (v/v) mixture of 10% aq. Na.sub.2S.sub.2O.sub.3 and sat. aq. NaHCO.sub.3 (20 ml). The aqueous phase was extracted with CH.sub.2Cl.sub.2 (330 ml), the combined organic fractions were dried over Na.sub.2SO.sub.4 and concentrated. The residue was purified by flash chromatography (EtOAc/hexanes 1:1 to 2:1) to give glycosyl phosphate 9* (218 mg, 0.272 mmol, 89%, 10:1 / as clear oil. Analytical data of 9*: HRMS (ESI) calcd for C.sub.44H.sub.51O.sub.12P (M+Na).sup.+825.3015. found 825.3020 m/z. Analytical data of 9*: HRMS (ESI) calcd for C.sub.44H.sub.51O.sub.12P (M+Na).sup.+825.3015. found 825.2970 m/z.

Example 9

Methyl (2-O-benzyl-3,4-O-endo-benzylidene-/-D-galactopyranosyl)uronate-(11)-2-(benzylthio)ethanol (10*)

(32) ##STR00110##

(33) Thioglycoside 5* (102 mg, 0.237 mmol), 2-(benzylthio)ethanol 11* (60 mg, 0.355 mmol) and TTBPy. (117 mg, 0.474 mmol) were co-evaporated with anh. toluene (310 ml) and kept under high vacuum for 30 min. The mixture was dissolved in THF (4.8 ml) and stirred over activated molecular sieves (3 ) for 30 min at room temperature. The solution was cooled to 0 C. and treated with DMTST (92 mg, 0.355 mmol in 0.2 ml dry CH.sub.2Cl.sub.2). The reaction was warmed to room temperature and stirred for 2 h at that temperature. The reaction was quenched with a 1:1 (v/v) MeOH/Et.sub.3N mixture (0.1 ml) and concentrated. The residue was purified by flash chromatography (EtOAc/hexanes/Et.sub.3N 0:1:0.01 to 30:70:0.01 to 45:55:0.01) to give thioether 10* (59 mg, 0.110 mmol, 46%) as a clear oil, along with the corresponding -isomer 10* (35 mg, 0.065 mmol, 27%). Analytical data for 10*: HRMS (ESI) calcd for C.sub.30H.sub.32O.sub.7S (M+Na).sup.+559.1766. found 559.1731 m/z.

Example 10

Methyl (2,4-di-O-benzyl--D-galactopyranosid)uronate-(11)-2-(benzylthio)ethanol (12*)

(34) ##STR00111##

(35) To a stirred solution of acetal 10*a (100.0 mg, 0.186 mmol) in dry THF (5.3 ml) was added first borane trimethylamine complex (57.4 mg, 0.745 mmol) and then aluminium chloride (149 mg, 1.118 mmol) at room temperature. The mixture was stirred for 4.5 h. The reaction was quenched by addition of water (10 ml) and 1 M aq. HCl (5 ml). The mixture was extracted with EtOAc (310 ml) and the combined organic fractions were dried over Na.sub.2SO.sub.4 and concentrated. The residue was purified by flash chromatography (EtOAc/hexanes 2:5 to 1:1) to give alcohol 12* (70.0 mg, 0.13 mmol, 70%) as a clear oil. HRMS (ESI) calcd for C.sub.30H.sub.34O.sub.7S (M+Na).sup.+561.1923. found 561.1879 m/z.

Example 11

Methyl (2,3-di-O-benzyl--D-galactopyranosyl)uronate-(13)-methyl (2,4-di-O-benzyl--D-galactopyranosyl)uronate-(13)-(2-(benzylthio)ethanol (13*)

(36) ##STR00112##

(37) Alcohol 12* (90 mg, 0.166 mmol) and glycosyl phosphate 9* (208 mg, 0.259 mmol) were co-evaporated with dry toluene (310 ml) and kept under high vacuum for 1 h. The mixture was dissolved in dry CH.sub.2Cl.sub.2 (3.3 ml) and stirred over activated molecular sieves (3 -AW) for 30 min at room temperature. The solution was cooled to 0 C. and treated dropwise with TBSOTf (0.133 mmol in 0.2 ml dry CH.sub.2Cl.sub.2). The solution was warmed to room temperature and stirred for 20 h. The reaction was diluted with CH.sub.2Cl.sub.2 (10 ml) and quenched with a 1:1 (v/v) MeOH/pyridine mixture (0.2 ml). The solution was filtered through Celite and concentrated. The crude product was filtered through a short plug of silica gel (EtOAc/hexanes 1:1) to give the intermediate disaccharide mixture (150 mg, 0.133 mmol, 80%, 3:1 /) as a clear oil.

(38) To a stirred solution of the carbonate mixture (150 mg) in CH.sub.2Cl.sub.2 (2.6 ml) was added at room temperature triethylamine (1.1 ml, 7.96 mmol). The reaction was stirred for 3 h at that temperature and co-evaporated with toluene (210 ml). The residue was purified by flash chromatography (EtOAc/hexanes 1:6 to 2:3 to 1:1) to give alcohol 13* (62 mg, 0.068 mmol, 51%) along with the corresponding -anomer (20 mg, 0.022 mmol, 17%). HRMS (ESI) calcd for C.sub.51H.sub.56O.sub.13S (M+Na).sup.+931.3339. found 931.3340 m/z.

Example 12

2-Azido-4-(benzyloxycarbonyl)amino-3-O-levulinoyl-2,4,6-trideoxy--D-galactopyranosyl-(14)-methyl (2,3-di-O-benzyl--D-galactopyranosyl)uronate-(13)-methyl (2,4-di-O-benzyl--D-galactopyranosyl)uronate-(11)-2-(benzylthio)ethanol (14*)

(39) ##STR00113##

(40) Alcohol 13* (65 mg, 0.062 mmol) and glycosyl phosphate 2* (61 mg, 0.100 mmol) were co-evaporated with dry toluene (310 ml) and kept under high vacuum for 30 min. The mixture was dissolved in CH.sub.2Cl.sub.2 (2.1 ml) and stirred over activated molecular sieves (4 -AW) for 1 h at room temperature. The solution was then cooled to 0 C. and treated with TMSOTf (17 l, 0.093 mmol in 0.2 ml dry CH.sub.2Cl.sub.2). The mixture was allowed to stir for 3 h at 0 C., when TLC (EtOAc/hexanes 2:3) indicated complete consumption of the acceptor. The reaction was quenched with a 1:1 (v/v) MeOH/NEt.sub.3 mixture (0.5 ml), diluted with CH.sub.2Cl.sub.2 (20 ml) and filtered through Celite. The crude product was purified by flash chromatography (EtOAc/hexanes 1:2 to 1:1) to give trisaccharide 14* (69 mg, 0.053 mmol, 85%) as a clear oil. HRMS (ESI) calcd. for C.sub.70H.sub.78N.sub.4O.sub.19S (M+Na).sup.+ 1333.4879. found 1333.4911 m/z.

Example 13

2-Azido-4-(benzyloxycarbonyl)amino-2,4,6-trideoxy--D-galactopyranosyl-(14)-methyl (2,3-di-O-benzyl--D-galactopyranosyl)uronate-(13)-methyl (2,4-di-O-benzyl--D-galactopyranosyl)uronate-(11)-2-(benzylthio)ethanol (15*)

(41) ##STR00114##

(42) To a stirred solution of levulinoyl ester 14* (30 mg, 0.023 mmol) in dry CH.sub.2Cl.sub.2 (1.0 ml) was added at room temperature first a mixture of pyridine (56 l, 0.692 mmol) and acetic acid (37 l, 0.646 mmol), and then hydrazine hydrate (2 l, 0.041 mmol). The mixture was allowed to stir for 4 h at room temperature, diluted with EtOAc (2 ml), quenched with acetone (0.1 ml) and poured into water (15 ml). The aqueous phase was extracted with EtOAc (410 ml), and the combined organic extracts were dried over Na.sub.2SO.sub.4 and concentrated under reduced pressure. The residue was purified by flash chromatography (EtOAc/hexanes 0:1 to 1:2 to 2:3) to give alcohol 15* (28 mg, 0.023 mmol, 100%) as a clear oil. HRMS (ESI) calcd. for C.sub.65H.sub.72N.sub.4O.sub.17S (M+Na).sup.+ 1235.4511. found 1235.4539 m/z.

Example 14

2-Azido-4-(benzyloxycarbonyl)amino-3-O-benzyloxymethyl-2,4,6-trideoxy--D-galactopyranosyl-(14)-methyl (2,3-di-O-benzyl--D-galactopyranosyl)uronate-(13)-methyl (2,4-di-O-benzyl--D-galactopyranosyl)uronate-(11)-2-(benzylthio)ethanol (16*)

(43) ##STR00115##

(44) Alcohol 15* (8.6 mg, 7.1 mol), benzyloxymethyl thiocyclohexane (79 mg, 0.354 mmol) and TTBPy. (105 mg, 0.425 mmol) were coevaproated with dry toluene (310 ml) and kept under high vacuum for 30 min. The mixture was dissolved in dry CH.sub.2Cl.sub.2 (0.4 ml) and stirred over activated molecular sieves (3 ) for 30 min at room temperature. The mixture was cooled to 0 C. and DMTST (7.1 mg, 0.18 mmol in 0.1 ml CH.sub.2Cl.sub.2) was added dropwise over 45 min, while the reaction temperature was kept below 10 C. The reaction was stirred for another 45 min, quenched by addition of a 1:1 (v/v) MeOH/Et.sub.3N mixture and concentrated. The residue was purified by flash chromatography (EtOAc/hexanes 1:10 to 1:2) to give acetal 16* (6.0 mg, 4.5 mol, 64%) as a clear oil. HRMS (ESI) calcd for C.sub.73H.sub.80N.sub.4O.sub.18S (M+Na).sup.+1355.5086. found 1355.5071 m/z.

Example 15

2-Acetamido-4-(benzyloxycarbonyl)amino-3-O-benzyloxymethyl-2,4,6-trideoxy--D-galactopyranosyl-(14)-methyl (2,3-di-O-benzyl--D-galactopyranosyl)uronate-(13)-methyl (2,4-di-O-benzyl--D-galactopyranosyl)uronate-(11)-2-(benzylthio)ethanol (17*)

(45) ##STR00116##

(46) To a stirred solution of azide 16* (14.0 mg, 10.5 mol) in dry pyridine (0.35 ml) was added at 0 C. thioacetic acid (0.35 ml). The mixture was warmed to room temperature and stirred for 24 h at that temperature. The solution was co-evaporated with toluene (25 ml) and the residue was purified by flash chromatography (EtOAc/hexanes 1:10 to acetone/hexanes 1:7 to 1:5 to 1:3) to give acetamide 17* (9.4 mg, 7.0 mol, 66%) as a white solid. HRMS (ESI) calcd for C.sub.75H.sub.84N.sub.2O.sub.19S (M+Na).sup.+1371.5281. found 1371.5314 m/z.

Example 16

2,2-Dithiobis[2-acetamido-4-amino-2,4,6-trideoxy--D-galactopyranosyl-(14)--D-galactopyranosyluronate-(13)--D-galactopyranosyluronate-(11)-1-ethanol] (18*)

(47) ##STR00117##

(48) To a stirred solution of diester 17* in THF (4.0 ml) and MeOH (0.8 ml) was added at 0 C. a 1 M solution of NaOH in water (1.5 ml). The reaction was slowly warmed to room temperature and stirred for 16 h. The reaction was diluted with EtOAc (5 ml) and acidified to pH 4 with 0.5 M aq. NaHSO.sub.4. After separation, the aqueous fraction was extracted with EtOAc (810 ml), the combined organic fractions were dried over Na.sub.2SO.sub.4 and concentrated to give the intermediate diacid as a white solid.

(49) To a stirring solution of liquid ammonia (5 ml) was added at 78 C. a solution of the crude diacid in THF (1.5 ml). The mixture was treated with tBuOH (0.5 ml) and lumps of freshly cut sodium (45 mg) were added until a deeply blue color persisted. The reaction was stirred at 78 C. for 45 min and quenched by addition of solid ammonium acetate (200 mg). The solution was warmed to room temperature under a stream of argon and co-evaporated with MeOH (210 ml) and water (25 ml). The residue was left under air for 16 h, purified by size exclusion chromatography (Sephadex G-25, 1:1 MeOH/5 mM aq. NH.sub.4OAc) and lyophilized repeatedly to give disulfide 18* (1.4 mg, 1.65 mol, 32%) as a white solid. HRMS (MALDI) calcd for C.sub.44H.sub.70N.sub.4O.sub.32S.sub.2(M-H.sup.+) 1229.3330. found 1229.3342 m/z.

Example 17

Methyl (ethyl 2,3-O-benzyl-4-O-levulinoyl-1-thio--D-galactopyranosyl)uronate (19*)

(50) ##STR00118##

(51) To a stirred solution of alcohol 7* (94 mg, 0.217 mmol) in CH.sub.2Cl.sub.2 (1.9 mL) were added at room temperature levulinic acid (386 mg, 3.26 mmol), DCC (673 mg, 3.26 mmol) and pyridine (0.26 mL, 3.26 mmol). The mixture was stirred at that temperature for 35 h, diluted with CH.sub.2Cl.sub.2 (5 mL) and filtered through Celite. The mixture was concentrated, the residue was dissolved in a minimal volume of CH.sub.2Cl.sub.2 (1-3 mL) and filtered through cotton wool. The same procedure was repeated 3 times. The residue was purified by flash chromatography (EtOAc/toluene 1:1) to give ester 19* (91 mg, 0.171 mmol, 79%) as a slightly yellow oil. HRMS (ESI) calcd for C.sub.28H.sub.34O.sub.8S (M+Na).sup.+ 553.1872. found 553.1872 m/z.

Example 18

Methyl (2,3-di-O-benzyl--D-galactopyranosid)uronate-(11)-6-(benzylthio)hexanol (20*)

(52) ##STR00119##

(53) Thioglycoside 19* (87 mg, 0.164 mmol), 6-(benzylthio)hexanol 21* (85 mg, 0.379 mmol) and TTBPy. (97 mg, 0.392 mmol) were co-evaporated with anh. toluene (310 ml) and kept under high vacuum for 30 min. The mixture was dissolved in Et.sub.2O (2.5 ml) and CH.sub.2Cl.sub.2 (0.83 ml) and stirred over activated molecular sieves (3 ) for 30 min at room temperature. The solution was cooled to 0 C. and treated with DMTST (63.5 mg, 0.246 mmol). The reaction was warmed to room temperature and stirred for 8 h at that temperature. The reaction was quenched with a 1:1 (v/v) mixture of MeOH and triethylamine (0.1 ml) and concentrated. The residue was purified by flash chromatography (EtOAc/CH.sub.2Cl.sub.2/hexanes 0:0:1 to 1:2:1) to give the corresponding glycosides (60 mg) as an inseparable / mixture.

(54) To a stirred solution of the glycoside mixture in dry CH.sub.2Cl.sub.2 (2.2 ml) were added at room temperature first a mixture of pyridine (195 l, 2.411 mmol) and acetic acid (137 l, 2.393 mmol), and then hydrazine hydrate (5.9 l, 0.121 mmol). The mixture was stirred for 2 h at that temperature, diluted with EtOAc (2 ml), quenched with acetone (0.1 ml) and poured into water (15 ml). The aqueous phase was extracted with EtOAc (410 ml), the combined organic extracts were dried over Na.sub.2SO.sub.4 and concentrated. The residue was purified by flash chromatography (EtOAc/hexanes 1:2) to give alcohol 20* (29 mg, 0.049 mmol, 30% over two steps) as a clear oil, along with the corresponding 1-isomer (22 mg, 0.037 mmol, 22%). HRMS (ESI) calcd. for C.sub.34H.sub.42O.sub.7S (M+Na).sup.+ 617.2544. found 617.2542 m/z.

Example 19

6,6-Dithiobis[-D-galactopyranosyluronate-(11)-1-hexanol] (22*)

(55) ##STR00120##

(56) To a stirred solution of ester 20* (10 mg, 0.017 mmol) in THF (1.0 ml) and MeOH (0.5 ml) was added at 0 C. a 1 M solution of NaOH in water (0.8 ml). The reaction was slowly warmed to room temperature and stirred for 16 h. The reaction was diluted with EtOAc (5 ml) and water (5 ml) and acidified to pH 4 with 0.5 M aq. NaHSO.sub.4. After separation, the aqueous fraction was extracted with EtOAc (85 ml), the combined organic fractions were dried over Na.sub.2SO.sub.4 and concentrated to give the intermediate acid as a white solid.

(57) To a stirred solution of liquid ammonia (8 ml) was added at 78 C. a solution of the crude diacid in THF (2 ml). The mixture was treated with tBuOH (0.4 ml) and lumps of freshly cut sodium (45 mg) were added until a deeply blue color persisted. The reaction was stirred at 78 C. for 45 min and quenched by addition of solid ammonium acetate (100 mg). The solution was warmed to room temperature under a stream of argon and co-evaporated with MeOH (210 ml) and water (25 ml). The residue was left under air for 16 h, purified by size exclusion chromatography (Sephadex G-25, 9:1 MeOH/5 mM aq. NH.sub.4OAc) and lyophilized repeatedly to give disulfide 22* (3.1 mg, 5.1 mol, 60% over two steps) as a white solid. HRMS (MALDI) calcd for C.sub.24H.sub.42O.sub.14S.sub.2 (M-H.sup.+) 617.1938. found 617.1954 m/z.

Example 20

2-Acetamido-4-(benzyloxycarbonyl)amino-3-O-levulinoyl-2,4,6-trideoxy--D-galactopyranosyl-(11)-6-(benzylthio)hexanol (23*)

(58) ##STR00121##

(59) 6-(Benzylthio)hexanol 21* (29 mg, 0.171 mmol) and glycosyl phosphate 2* (70 mg, 0.114 mmol) were co-evaporated with dry toluene (310 ml) and kept under high vacuum for 30 min. The mixture was dissolved in CH.sub.2Cl.sub.2 (1.8 ml) and stirred over activated molecular sieves (4 -AW) for 1 h at room temperature. The solution was then cooled to 0 C. and treated with TMSOTf (31 l, 0.171 mmol in 0.2 ml dry CH.sub.2Cl.sub.2). The mixture was stirred for 3 h at that temperature, quenched with a 1:1 (v/v) mixture of MeOH and triethylamine (0.5 ml), diluted with CH.sub.2Cl.sub.2 (20 ml) and filtered through Celite. The residue was purified by flash chromatography (EtOAc/hexanes 2:3 to 3:2) to give the corresponding glycosides (57 mg) as an inseparable / mixture.

(60) To a stirred solution of the glycoside mixture in dry pyridine (0.9 ml) was added at 0 C. thioacetic acid (0.9 ml). The mixture was warmed to room temperature and stirred for 24 h at that temperature. The solution was co-evaporated with toluene (25 ml) and the residue was purified by flash chromatography (EtOAc/hexanes 1:2 to 2:1 to 6:1) to give acetamide 23* (22 mg, 0.034 mmol, 29% over two steps) as a white solid, along with the corresponding -isomer (21.6 mg, 0.034 mmol, 29%). HRMS (ESI) calcd for C.sub.34H.sub.46N.sub.2O.sub.8S (M+Na).sup.+ 665.2872. found 665.2865 m/z.

Example 21

6,6-Dithiobis[2-acetamido-4-amino-2,4,6-trideoxy--D-galactopyranosyl-(11)-1-hexanol] (24*)

(61) ##STR00122##

(62) To a stirred solution of ester 23* (10 mg, 0.016 mmol) in dry CH.sub.2Cl.sub.2 (1.0 ml) were added at room temperature first a mixture of pyridine (38 l, 0.467 mmol) and acetic acid (24.9 l, 0.436 mmol), and then hydrazine hydrate (1.0 l, 0.020 mmol). The mixture was stirred for 2 h at that temperature, quenched with acetone (0.1 ml) and purified by size exclusion chromatography (Sephadex LH-20, CH.sub.2Cl.sub.2/MeOH 2:1) to give the corresponding alcohol as a clear oil.

(63) To a stirred solution of liquid ammonia (5 ml) was added at 78 C. a solution of the intermediate alcohol in THF (1.2 ml). The mixture was treated with tBuOH (0.5 ml) and lumps of freshly cut sodium (80 mg) were added until a deeply blue color persisted. The reaction was stirred at 78 C. for 45 min and quenched by addition of solid ammonium acetate (100 mg). The solution was warmed to room temperature under a stream of argon and co-evaporated with MeOH (210 ml) and water (25 ml). The residue was left under air for 16 h, purified by size exclusion chromatography (Sephadex G-25, 9:1 MeOH/5 mM aq. NH.sub.4OAc) and lyophilized repeatedly to give disulfide 24* (1.7 mg, 2.7 mol, 33% over two steps) as a white solid. HRMS (ESI) calcd. for C.sub.28H.sub.54N.sub.4O.sub.8S.sub.2(M+Na).sup.+ 661.3281. found 661.3306 m/z.

Example 22

2-Acetamido-4-(benzyloxycarbonyl)amino-3-O-levulinoyl-2,4,6-trideoxy--D-galactopyranosyl-(11)-2-(benzylthio)ethanol (25*)

(64) ##STR00123##

(65) 2-(benzylthio)ethanol 11* (71 mg, 0.421 mmol) and glycosyl phosphate 2* (171 mg, 0.281 mmol) were co-evaporated with dry toluene (310 ml) and kept under high vacuum for 30 min. The mixture was dissolved in CH.sub.2Cl.sub.2 (1.8 ml) and stirred over activated molecular sieves (4 -AW) for 1 h at room temperature. The solution was then cooled to 40 C. and treated with TMSOTf (56 l, 0.309 mmol in 0.2 ml dry CH.sub.2Cl.sub.2). The mixture was slowly warmed to 0 C. (2 h), quenched with a 1:1 (v/v) mixture of MeOH and triethylamine (0.5 ml), diluted with CH.sub.2Cl.sub.2 (20 ml), filtered through Celite and concentrated. The residue was purified by flash chromatography (EtOAc/hexanes 1:3 to 1:1) to give the corresponding -glycoside (55 mg, 0.096 mmol, 34%) along with the corresponding -glycoside (22 mg, 0.039 mmol, 14%). To a stirred solution of the intermediate -glycoside (40 mg, 0.070 mmol) in dry pyridine (0.4 ml) was added at 0 C. thioacetic acid (0.4 ml). The mixture was warmed to room temperature and stirred for 24 h at that temperature. The solution was co-evaporated with toluene (25 ml) and the residue was purified by flash chromatography (EtOAc/hexanes 1:3 to acetone/hexanes 1:2 to 2:3) to give acetamide 25* (31 mg, 0.053 mmol, 76%) as a white solid. HRMS (ESI) calcd for C.sub.30H.sub.38N.sub.2O.sub.8S (M+Na).sup.+609.2246. found 609.2256 m/z.

Example 23

6,6-Dithiobis[2-acetamido-4-amino-2,4,6-trideoxy--D-galactopyranosyl-(11)-1-ethanol] (26*)

(66) ##STR00124##

(67) To a stirred solution of ester 25* (20.7 mg, 0.035 mmol) in dry CH.sub.2Cl.sub.2 (3.0 ml) were added at room temperature first a mixture of pyridine (86 l, 1.058 mmol) and acetic acid (57 l, 0.988 mmol), and then hydrazine hydrate (3.4 l, 0.071 mmol). The mixture was stirred for 5 h at that temperature, diluted with EtOAc (2 ml), quenched with acetone (0.1 ml) and poured into water (10 ml). The aqueous phase was extracted with EtOAc (45 ml), the combined organic fractions were dried over Na.sub.2SO.sub.4 and concentrated. The residue was purified by flash chromatography (acetone/hexanes 1:1) to give the intermediate alcohol (17.5 mg) as a white solid.

(68) To a stirred solution of liquid ammonia (6 ml) was added at 78 C. a solution of the intermediate alcohol in THF (1.5 ml). The mixture was treated with tBuOH (0.5 ml) and lumps of freshly cut sodium (45 mg) were added until a deeply blue color persisted. The reaction was stirred at 78 C. for 45 min and quenched by addition of solid ammonium acetate (100 mg). The solution was warmed to room temperature under a stream of argon and co-evaporated with MeOH (210 ml) and water (25 ml). The residue was left under air for 16 h, purified by size exclusion chromatography (Sephadex G-25, 1:10 MeOH/5 mM aq. NH.sub.4OAc) and lyophilized repeatedly to give disulfide 26* as the diacetate salt (7.91 mg, 12.3 mol, 70% over two steps) as a white solid. HRMS (ESI) calcd. for C.sub.20H.sub.38N.sub.4O.sub.8S.sub.2(M+Na).sup.+ 549.2029. found 549.2086 m/z.

Example 24

2,2-Dithiobis[-D-galactopyranosyluronate-(13)--D-galactopyranosyluronate-(11)-1-ethanol] (27*)

(69) ##STR00125##

(70) To a stirred solution of ester 13* (8.6 mg, 9.5 mol) in THF (0.6 ml) and MeOH (0.3 ml) was added at 0 C. a 1 M solution of NaOH in water (0.5 ml). The reaction was slowly warmed to room temperature and stirred for 16 h. The reaction was diluted with EtOAc (5 ml) and water (5 ml) and acidified to pH 4 with 0.5 M aq. NaHSO.sub.4. After separation, the aqueous fraction was extracted with EtOAc (85 ml), the combined organic fractions were dried over Na.sub.2SO.sub.4 and concentrated to give the intermediate diacid as a white solid.

(71) To a stirred solution of liquid ammonia (6 ml) was added at 78 C. a solution of the crude diacid in THF (1.5 ml). The mixture was treated with tBuOH (0.4 ml) and lumps of freshly cut sodium (75 mg) were added until a deeply blue color persisted. The reaction was stirred at 78 C. for 45 min and quenched by addition of solid ammonium acetate (100 mg). The solution was warmed to room temperature under a stream of argon and co-evaporated with MeOH (210 ml) and water (25 ml). The residue was left under air for 16 h, purified by size exclusion chromatography (Sephadex G-25, 1:9 MeOH/5 mM aq. NH.sub.4OAc) and lyophilized repeatedly to give disulfide 27* (2.5 mg, 2.9 mol, 61% over two steps) as a white solid. HRMS (MALDI) calcd for C.sub.28H.sub.42O.sub.26S.sub.2(M-H.sup.+) 901.0966. found 901.0981 m/z.

Example 25

Synthesis of GlycoconjugatesConjugation of Saccharides of General Formula (I) to CRM197

(72) To a stirred solution of CRM.sub.197 (1 mg, 17.2 nmol) in 0.1 M sodium phosphate buffer (NaPi) pH 7.4 (1 ml) was added at room temperature a solution of succinimidyl-3-(bromoacetamido)propionate (SBAP) (264 g, 863 nmol) in DMF (20 l). The mixture was stirred for 1 h at that temperature, and concentrated using membrane filtration (Amicon Ultra centrifuge membranes, 10 kDa cut-off). The protein solution was diluted with 0.1 M NaPi pH 7.4 and concentrated again. This process was repeated three times and the solution was diluted to 1 ml using 0.1 M NaPi pH 7.4. The intermediate of general formula (II) (690 mmol) in 120 l0.1 M NaPi pH 7.4 was treated at room temperature with tris(2-carboxyethyl)phosphine (TCEP) (690 mmol), left for 1 h at that temperature under an argon atmosphere and added to the solution of the bromoacetamido-modified CRM.sub.197 protein at room temperature. The mixture was left at room temperature for 2 h and then at 4 C. for 16 h, and purified using membrane filtration (see above). The purified glycoconjugate in 0.1 M NaPi pH 7.4 (1 ml) was then treated at room temperature with L-cysteine (417 g, 3.45 mol) in 100 l water. The mixture was left for 2 h at that temperature and purified by membrane filtration. Incorporation of the saccharide of general formula (I) into the glycoconjugate was assessed by MALDI-TOF-MS, SDS-PAGE and size exclusion chromatography with right angle light scattering detection (SEC-RALS).

Example 26

Synthesis of Glycoconiugatesin Flow Conjugation of the Saccharides of General Formula (I) to a Glycosphingolipid with Immunomodulatory Properties

(73) By using a photochemical flow reactor (Chem. Eur. J. 2013, 19, 3090) that was fitted with a loop of Teflon AF2400 tubing (566 L), a solution of saccharide of general formula (I) (1.5 equiv.) in water (300 L) was reacted with pentenyl modified (2S,3S,4R)-1-(-D-galactopyranosyl)-2-hexacosanoylaminooctadecane-3,4-diol (1 equiv.) in water (300 L) and AcOH (8 L; residence time: 10 min, flow rate: 28.3 L/min.sup.1 per syringe). The reactor output was lyophilized and the crude material was purified using size exclusion chromatography (Sephadex-G25, 5% EtOH in water, 10 mm150 mm) to yield the glycoconjugate of saccharide of general formula (I) covalently linked to the modified (2S,3S,4R)-1-(-D-galactopyranosyl)-2-hexacosanoylaminooctadecane-3,4-diol as white solid.

Example 27

Synthesis of glycoconiugatesConjugation of 2-acetamido-4-amino-2,4,6-trideoxy--D-galactopyranosyl-(14)-(-D-galactopyranosyl)uronate-(13)-(-D-galactopyranosyl)uronate-(11)-2-(thio)ethanol to BSA

(74) To a stirred solution of BSA (0.5 mg, 7.6 nmol) in 0.1 M sodium phosphate buffer (NaPi) pH 7.4 (1 mL) was added at room temperature a solution of N-succinimidyl-3-(bromoacetamido)propionate (SBAP) (89 g, 290 nmol) in DMF (20 L). The mixture was stirred for 1 h at room temperature, and concentrated using membrane filtration (Amicon 0.5 mL Ultra centrifuge membranes, 10 kDa cut-off). The protein solution was diluted with 0.1 M NaPi pH 7.4 and concentrated again. This process was repeated three times and the solution was diluted to 0.5 mL using water. 20 L were taken for analysis, and the protein solution was re-buffered to 0.1 M NaPi pH 7.4 using membrane filtration. Disulfide 18* (140 g, 228 nmol resp. to the monomer) in 120 L 0.1 M NaPi pH 7.4 was treated at room temperature with tris(2-carboxyethyl)phosphine (TCEP) (250 nmol), left for 1 h at that temperature under an argon atmosphere and added to the solution of the activated protein at room temperature. The mixture was left at 4 C. for 16 h, and purified using membrane filtration (see above). After washing with water and diluting to 0.5 mL, another analytical sample (20 L) was taken, and the solution was re-buffered. The purified glycoconjugate in 0.1 M NaPi pH 7.4 (0.5 mL) was then treated at room temperature with L-cysteine (417 g, 3.45 mol) in 100 l water. The mixture was left for 2 h at that temperature and purified by membrane filtration. Incorporation of 2-acetamido-4-amino-2,4,6-trideoxy--D-galactopyranosyl-(1.fwdarw.4)-(-D-galactopyranosyl)uronate-(1.fwdarw.3)-(-D-galactopyranosyl)uronate-(1.fwdarw.1)-2-(thio)ethanol into the glycoconjugate was assessed by MALDI-TOF-MS (positive mode):

(75) Molecular Weight Measured:

(76) BSA: 66341 m/z.

(77) BSA-SBAP conjugate: 68316 m/z (incorporation of approximately 10 SBAP groups).

(78) BSA-SBAP glycoconjugate: 69101 m/z (incorporation of approximately 1.3 molecules of 2-acetamido-4-amino-2,4,6-trideoxy--D-galactopyranosyl-(1.fwdarw.4)-(-D-galactopyranosyl)uronate-(1.fwdarw.3)-(-D-galactopyranosyl)uronate-(1.fwdarw.1)-2-(thio)ethanol).

(79) BSA-SBAP glycoconjugate after quenching with L-cysteine: 72074 m/z (incorporation of approximatively 24.5 L-cysteine molecules).

Example 28

Synthesis of glycoconiugatesConjugation of 2-acetamido-4-amino-2,4,6-trideoxy--D-galactopyranosyl-(14)-(-D-galactopyranosyl)uronate-(13)-(-D-galactopyranosyl)uronate-(11)-2-(thio)ethanol to BSA

(80) To a stirred solution of BSA (0.5 mg, 7.6 nmol) in 0.1 M sodium phosphate buffer (NaPi) pH 7.4 (1 mL), a solution of N-Succinimidyl-3-maleimidopropionate (101 g, 380 nmol) in DMF (20 L) was added at room temperature. The mixture was stirred for 1 h at room temperature, and concentrated using membrane filtration (Amicon 0.5 mL Ultra centrifuge membranes, 10 kDa cut-off). The protein solution was diluted with 0.1 M NaPi pH 7.4 and concentrated again. This process was repeated three times and the solution was diluted to 0.5 mL using water. 20 L were taken for analysis, and the protein solution was re-buffered to 0.1 M NaPi pH 7.4 using membrane filtration. Disulfide 18* (140 g, 228 nmol resp. to the monomer) in 120 l 0.1 M NaPi pH 7.4 was treated at room temperature with tris(2-carboxyethyl)phosphine (TCEP) (250 nmol), left for 1 h at that temperature under an argon atmosphere and added to the solution of the activated protein at room temperature. The mixture was left at 4 C. for 16 h, and purified using membrane filtration (see above). After washing with water and diluting to 0.5 mL, another analytical sample (20 L) was taken, and the solution was re-buffered. The purified glycoconjugate in 0.1 M NaPi pH 7.4 (0.5 mL) was then treated at room temperature with L-cysteine (417 g, 3.45 mol) in 100 l water. The mixture was left for 2 h at that temperature and purified by membrane filtration. Incorporation of glycan into the glycoconjugate was assessed by MALDI-TOF-MS (positive mode):

(81) Molecular Weight Measured:

(82) BSA: 66341 m/z;

(83) BSA-maleimide conjugate: 69254 m/z (incorporation of approximately 19 maleimide groups);

(84) BSA-maleimide glycoconjugate: 71340 m/z (incorporation of approximately 3.4 molecules of 2-acetamido-4-amino-2,4,6-trideoxy--D-galactopyranosyl-(1.fwdarw.4)-(-D-galactopyranosyl)uronate-(1.fwdarw.3)-(-D-galactopyranosyl)uronate-(1.fwdarw.1)-2-(thio)ethanol);

(85) BSA-maleimide glycoconjugate after quenching with L-cysteine: 72106 m/z (incorporation of approx. 6.3 L-cysteine molecules).

Example 29

Conjugation to a Solid SupportSynthesis of Microarrays Using GAPSII Slides

(86) Maleimide-functionalized microarrays were produced by submerging amine-coated slides (GAPS II slides, Corning) in 6-maleimidohexanoic acid N-hydroxysuccinimide ester (2 mM) in dry DMF with diisopropylethylamine (2.5% v/v) for 24 h at room temperature. Slides were washed three times with water and three times with ethanol, centrifuged to dryness, and stored under argon until spotting. Diluted saccharides of general formula (I) were printed onto the modified microarray slides at 0.4 nL per spot by an automatic piezoelectric arraying robot (Scienion, Berlin, Germany). For completion of the immobilization reaction, printed slides were stored for 24 h in a humidified chamber.

(87) Microarray slides were washed three times with water. Unreacted maleimide was quenched by submerging the slides in -mercaptoethanol (0.1%, v/v) in PBS for 1 h at room temperature. Slides were washed three times with water and with ethanol, centrifuged to dryness, and blocked with BSA (1%, w/v) in PBS for 1 h at room temperature. Blocked slides were washed (2PBS, 3water), centrifuged, and incubated with the sera dilutions.

Example 30

Conjugation to a Solid SupportSynthesis of Microarrays Using CodeLink NHS Slides

(88) CodeLink NHS slides were incubated for 24 h (1% w/v in PBS) at 4 C. Slides were incubated in blocking buffer (100 mM ethanolamine in 50 mM NaPi pH>9) for 30 min at room temperature, washed three times each with water and ethanol, and dried. Slides were then subjected to maleimide functionalization and printing (see Example 29).

Example 31

Binding Experiments Using the Microarrays Synthesized According to the Procedure Described at Examples 29 and 30

(89) Binding experiments were performed by incubating microarray slides coated with the saccharides of general formula (I) with either a rabbit anti-SP1 typing serum or human pneumococcal reference serum 007sp (pooled sera of 287 humans immunized with Pneumovax vaccine) in the dilutions indicated in the presence or absence of native SP1 polysaccharide, and using fluorescently labeled anti-rabbit or anti-human secondary antibodies.

Example 32

Assessment of the Immunogenicity of the Linker A and of the Interconnecting Molecule

(90) To check the immunogenicity of the linker A and of the interconnecting molecule used in preparing the glycoconjugate 1 according to the present invention i.e. a glycoconjugate containing

(91) a saccharide of general formula I presenting a linker A connected via an interconnecting molecule 1 to immunogenic carrier 1 such as: H(P).sub.n3(N).sub.n2-(M).sub.n3-O-A-S-rest interconnecting molecule 1-immunogenic carrier 1, three glycoconjugates need to be synthesized:

(92) Glycoconjugate 2: H(P).sub.n3(N).sub.n2-(M).sub.n3-O-A-S-rest interconnecting molecule 2-immunogenic carrier 2;

(93) Glycoconjugate 3: galactose-O-A-S-rest interconnecting molecule 1-immunogenic carrier 2;

(94) Glycoconjugate 4: galactose-O-A-S-rest interconnecting molecule 2-immunogenic carrier 2;

(95) The immunogenic carrier 2 has to be non-related to immunogenic carrier 1 used in immunization. For example, if CRM197 was used to prepare glycoconjugate 1, then BSA can be used as immunogenic carrier 2.

(96) The galactose was chosen for preparing glycoconjugate 3, as it is non related to the saccharides according to the present invention i.e. to H(P).sub.n3(N).sub.n2-(M).sub.n3-OH.

(97) The interconnecting molecule 2 used for the preparation of glycoconjugate 2 has to be non related to interconnecting molecule 1. For example, if Sulfo-GMBS was used as interconnecting molecule 1, then a non-related interconnecting molecule 2 would be Sulfo SIAB.

(98) The choice of immunogenic carrier 2, interconnecting molecule 2 and of non related saccharide are obvious for the person skilled in the art of synthesis of glycoconjugates.

(99) Protocol for ELISA:

(100) A 96-well-plate is coated with 50 l of the respective glycoconjugates (50 g/ml) in PBS for 1 h at 37 C. The plate is washed once with 100 l washing buffer (PBS+0.1% (v/v) Tween-20) and blocked using 200 l blocking solution (1% (w/v) BSA in PBS) for 1 h at 37 C. The plate is washed 3 with washing buffer and then incubated with dilutions of antiserum in blocking solution (50 l) for 16 h at 4 C. The plate is washed 3 and incubated with 50 l of an appropriate secondary antibody (e.g. Goat Anti-Mouse IgG H&L (HRP), abcam ab6789) diluted in blocking solution for 1 h at 37 C. The plate is washed 3 with washing buffer and incubated with ELISA substrate (e.g. ABTS from Pierce, No. 37615) according to the manufacturer's manual.

(101) Comparison of the optical density between the glycoconjugates applied will give a quantitative comparison between anti-linker-, anti-interconnecting molecule, and anti-saccharide immune responses.

Example 33

2-Azido-4-(benzyloxycarbonyl)amino-2,4,6-trideoxy--D-galactopyranosyl-(11)-6-(benzylthio)ethanol (28*)

(102) ##STR00126##

(103) To a stirred solution of the intermediate Lev ester 25* (17 mg, 0.03 mmol) in dry CH.sub.2Cl.sub.2 (1 mL) were added at room temperature first a mixture of pyridine (72 l, 0.894 mmol) and acetic acid (48 l, 0.834 mmol), and then hydrazine hydrate (3 l, 0.062 mmol). The mixture was stirred for 5 h at that temperature, diluted with EtOAc (2 ml), quenched with acetone (0.1 mL) and poured into water (10 mL). The aqueous phase was extracted with CH.sub.2Cl.sub.2 (45 ml), the combined organic fractions were dried over Na.sub.2SO.sub.4 and concentrated. The residue was purified by flash chromatography (EtOAc/hexanes 3:1) to give alcohol 28* (13 mg, 0.028 mmol, 92%) as a clear oil. HRMS (ESI) calcd. for C.sub.23H.sub.28N.sub.4O.sub.5S (M+Na).sup.+ 495.1678. found 495.1679 m/z.

Example 34

Methyl (2,3-di-O-benzyl--D-galactopyranosyl)uronate-(13)-2-azido-4-(benzyloxycarbonyl)amino-2,4,6-trideoxy--D-galactopyranosyl-(11)-2-(benzylthio)ethanol (29*)

(104) ##STR00127##

(105) Alcohol 28*(13 mg, 0.028 mmol), TTBPy (45, 0.138 mmol) and thioglycoside 19* (37 mg, 0.069 mmol) were co-evaporated with dry toluene (310 mL) and kept under high vacuum for 1 h. The mixture was dissolved in dry THF (1.5 mL) and stirred over activated molecular sieves (3 ) for 30 min at room temperature. The solution was cooled to 0 C. and treated dropwise with DMTST (17 mg, 0.069 mmol in 0.2 mL DCM) The mixture was warmed to room temperature and treated with an additional DMTST solution in DCM (2 equiv.) after 2 h. The reaction was stirred for 16 h and quenched with 1:1 (v/v) mixture of 10% aq. Na.sub.2S.sub.2O.sub.3 and sat. aq. NaHCO.sub.3 (5 mL). The mixture was extracted with CH.sub.2Cl.sub.2 (310 mL), dried over Na.sub.2SO.sub.4 and concentrated. The residue was purified by flash chromatography (EtOAc/hexanes 1:2) to give the intermediate disaccharide as a clear oil.

(106) To a stirred solution of the intermediate disaccharide in dry pyridine (0.2 ml) was added at 0 C. thioacetic acid (0.2 ml). The mixture was warmed to room temperature and stirred for 24 h at that temperature. The solution was co-evaporated with toluene (25 ml) and the residue was purified by flash chromatography (EtOAc/hexanes 1:3 to acetone/hexanes 1:2) to give the intermediate acetamide as a white foam.

(107) To a stirred solution of the intermediate acetamide in dry CH.sub.2Cl.sub.2 (0.6 mL) and MeOH (60 L) were added at room temperature first a mixture of pyridine (12 l, 0.16 mmol) and acetic acid (8 l, 0.15 mmol), and then hydrazine hydrate (1 l, 0.021 mmol). The mixture was stirred for 3 h at that temperature, diluted with CH.sub.2Cl.sub.2 (2 ml), quenched with acetone (0.1 mL) and poured into water (5 mL). The aqueous phase was extracted with CH.sub.2Cl.sub.2 (45 ml), the combined organic fractions were dried over Na.sub.2SO.sub.4 and concentrated. The residue was purified by flash chromatography (acetone/hexanes 0:1 to 1:1) to give acetamide 29* (2.7 mg, 3.14 mol, 21% over 3 steps based on recovered 28*) as a white foam. HRMS (ESI) calcd. for C.sub.46H.sub.54N.sub.2O.sub.12S (M+Na).sup.+881.3295. found 881.3286 m/z.

Example 35

2,2-Dithiobis[-D-galactopyranosyluronate-(13)-2-acetamido-4-amino-2,4,6-trideoxy--D-galactopyranosyl-(11)-1-ethanol] (30*)

(108) ##STR00128##

(109) To a stirred solution of ester 29* (2.7 mg, 3.14 mol) in THF (1 mL) and MeOH (0.25 mL) was added at 0 C. a 1 M solution of NaOH in water (0.4 mL). The reaction was slowly warmed to room temperature and stirred for 16 h. The reaction was diluted with EtOAc (5 ml) and water (5 ml) and acidified to pH 4 with 0.5 M aq. NaHSO.sub.4. After separation, the aqueous fraction was extracted with EtOAc (85 ml), the combined organic fractions were dried over Na.sub.2SO.sub.4 and concentrated to give the intermediate diacid as a white solid.

(110) To a stirred solution of liquid ammonia (10 ml) was added at 78 C. a solution of the crude diacid in THF (1.5 ml). The mixture was treated with tBuOH (0.4 ml) and lumps of freshly cut sodium (80 mg) were added until a deeply blue color persisted. The reaction was stirred at 78 C. for 45 min and quenched by addition of solid ammonium acetate (100 mg). The solution was warmed to room temperature under a stream of argon and co-evaporated with MeOH (210 ml) and water (25 ml). The residue was left under air for 16 h, purified by size exclusion chromatography (Sephadex G-25, 1:3 MeOH/5 mM aq. NH.sub.4OAc) and lyophilized repeatedly to give disulfide 30* (1.15 mg, 2.61 mol, 83% over two steps) as a white solid:

(111) .sup.1H NMR (600 MHz, D.sub.2O) 5.16 (d, J=2.0 Hz, 1H), 5.02 (d, J=3.2 Hz, 1H), 4.49 (d, J=5.9 Hz, 1H), 4.44-4.35 (m, 1H), 4.34-4.20 (m, 2H), 4.15-4.04 (m, 1H), 4.00-3.78 (m, 5H), 3.06 (t, J=5.6 Hz, 2H), 2.09 (s, 3H), 1.41 (d, J=6.6 Hz, 3H). LRMS calcd. for C.sub.32H.sub.54N.sub.4O.sub.20S.sub.2(M+2H).sup.2+ 440.4. found 440.2 m/z.

Example 36

General Procedures for Accessing the Compound of General Formula 3 According to the Present Invention

(112) ##STR00129##

Example 36.1

(113) ##STR00130##

(114) To a solution of isocyanate A12 (2.70 mmol) in 12 ml of CH.sub.2Cl.sub.2 was added the amine (1.05 eq, 2.83 mmol). The reaction mixture was stirred at ambient temperature for 12 hours and then concentrated in vacuo. The crude material was dissolved in Et.sub.2O followed by addition of hexane. The urea was then precipitated and filtered to get product.

(115) A mixture of p-methoxybenzyl ether protected urea compound (0.4 mmol) and 1,3,5-trimethoxybenzene (0.2 mmol) in anhydrous CH.sub.2Cl.sub.2 (3 mL) was added via a cannula to a solution of silver hexafluoroantimonate (20 mol, 5 mol %) in anhydrous CH.sub.2Cl.sub.2 (1 mL). The reaction mixture was heated to reflux until completion and filtered through a small pad of Celite with dichloromethane as eluent. Solvents were removed in vacuum, and the crude residue was purified by flash chromatography to get A10.

Example 36.2

(116) ##STR00131##

(117) wherein: R.sup.3 represents Me and R.sup.4 represents OH

(118) To a mixture of aldehyde A17 (1.0 mmol) and ketone A16 (1.0 mmol) in i-PrOH (100 iL) were added propionic acid (0.1 mmol, 10 mol %) and pyrrolidine (0.1 mmol, 10 mol %). The reaction mixture was stirred at 45 C. for 1-25 h. NaHCO.sub.3 was added, and the mixture was extracted with CH.sub.2Cl.sub.2 (3 5 mL). The combined extracts were washed with brine, dried (Na.sub.2SO.sub.4), and concentrated in vacuo. The residue was purified by flash chromatography to give intermediate unsaturated ketone A15.

(119) A 9.510.sup.3 M solution of intermediate unsaturated ketone A15 (0.05 mmol) in THF and recently prepared Stryker's reagent (0.025 mmol), were mixed together forming a homogeneous solution that was stirred at room temperature for 2 h. The reaction was quenched with saturated aq. NH.sub.4Cl. The mixture was stirred for 1 h. The reaction mixture was filtered, and the residue was washed with ethyl acetate. The organic phase was separated, and the aqueous phase was extracted with ethyl acetate. The combined organic phases were dried with MgSO.sub.4 and the solvent was removed under vacuum. The residue was purified by column chromatography on silica gel to give ketone A14.

(120) Neat tris-nonyloxy methyl titanium (10.1 mmol) was placed in a two-necked round bottom flask and subjected to Ar atmosphere. Ketone A14 (4.65 mmol) in THF was added and the mixture was stirred at room temperature for 30 min. Oleic acid (17.8 mmol) was added and the mixture was heated to 110 C. The product was concentrated and purified by flash chromatography to give the intermediate tertiary alcohol.

(121) To a solution of the PMB ether (0.1 mmol) in dichloroethane (5 mL), POCOl.sub.3 (0.5 mmol) was added and stirred at room temperature. After completion of the reaction, it was quenched in ice water and the organic layer was separated and the aqueous layer was extracted with dichloroethane (25 mL). Combined organic layer was washed with brine solution, dried (Na.sub.2SO.sub.4), concentrated under reduced pressure and the residue was purified by column chromatography (silica gel, EtOAc:Hexanes) to afford alcohol A13.

Example 36.3

(122) ##STR00132##

(123) An oven-dried, 250-mL, round-bottomed flask was charged with 1,3-propanedithiol (20 mmol, 1.1 equiv) and tetrabutylammonium iodide (0.40 mmol, 2.2 mol %) in dry THF (100 mL). The mixture was stirred at room temperature and sodium hydride (60% suspension in mineral oil, 20 mmol, 1.1 equiv) was added by portions. The resulting mixture was stirred for 30 min, then benzyl bromide (18 mmol) was added dropwise. The solution was stirred for 1 h at room temperature, then filtered on a frit funnel and concentrated under vacuum. The resulting crude oil was distilled under vacuum to afford the title compound as colorless oil A20.

(124) To a solution of A19 (0.11 mmol) in anhydrous DMF (2 mL) was added thiol A20 (0.22 mmol). The mixture was stirred at 25 C. for 18 h and then concentrated in vacuum. The residue was purified by flash chromatography to afford A18.

(125) A variety of dithiol derivatives of general formula A20 are commercially available.

Example 36.4

(126) ##STR00133##

(127) Freshly cut sodium metal (4.67 mmol) was dissolved in isopropanol (10 mL) and benzyl mercaptan (6.23 mmol) was added. A solution of 4-(bromomethyl)cyclopent-1-ene (1.55 mmol) in isopropanol (5 mL) was added and the solution was heated under reflux for 4 days. The solution was allowed to cool to room temperature, diluted with water (50 mL) and extracted with diethyl ether (350 mL). The combined organic extracts were washed with 0.1 M aq potassium hydroxide (250 mL), dried and evaporated to yield crude A25. Column chromatography on silica gel eluting with ethyl acetate/hexane afforded the title compound A25.

(128) A dry 50-mL flask equipped with a magnetic stirring bar, a septum inlet, an oil bubbler, and a reflux condenser was flushed with nitrogen. To the flask were added an alkene A25 (5.5 mmol) and dry THF (2.5 mL) and then a solution of 9-BBN (0.5 M solution in THF, 5.5 mmol) at 0 C. The mixture was warmed up slowly to room temperature and then stirred for 4-6 h to give a solution of B-alkyl-9-BBN A24.

(129) To the above borane solution of A24 were added DMF-THF (25 mL), PdCl.sub.2(dppf) (0.15 mmol), haloalkene (5 mmol), and powdered K.sub.3PO.sub.4 (6 mmol). The mixture was stirred for 8 h at 50 C. and then poured into water. The product was extracted with benzene, washed with water four times, and dried over MgSO.sub.4. Column chromatography on silica gel eluting with ethyl acetate/hexane afforded the title compound A22.

Example 36.5

(130) ##STR00134##

(131) To a solution of 1.1 equiv aldehyde A30 in dry methanol was dropwise added 1.3 equiv of amine A28 in dry methanol at 0 C. under an argon atmosphere. The mixture was stirred for an additional 10 min at room temperature and followed by the sequential addition of 1.0 equiv acid A27 in dry methanol and 1.1 equiv isocyanide A29. The reaction mixture was stirred for 24-48 h at room temperature. The resulting solution was diluted with dichloromethane and washed with 1 N HCl aqueous solution followed by saturated sodium bicarbonate aqueous solution and brine. The organic layer was dried over MgSO4, concentrated and purified by silica gel column chromatography (hexanes/ethyl acetate).

Example 36.6

(132) ##STR00135##

(133) Synthesis of A33:

(134) To the solution of azide A35 (0.03 mol) and ammonium chloride (0.07 mol) in ethyl alcohol (80 mL) and water (27 mL), zinc powder (0.04 mol) was added, the mixture was stirred vigorously at room temperature or at refluxing. After the reaction is over, ethyl acetate (200 mL) and aqueous ammonia (10 mL) was added. The mixture was filtered, and the filtrate was washed with brine, dried over anhydrous sodium sulfate and concentrated under reduced pressure.

(135) Synthesis of A31:

(136) Acid A32 (1 mmol) and amine A33 (1 mmol) were coupled with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) hydrochloride (1 mmol) in 3.5 mL methanol for 3 h at room temperature. The mixture was diluted with EtOAc (20 mL) and extracted with water (10 mL), 1 M aq. HCl (10 mL) and sat. aq. NaHCO.sub.3 (10 mL). The organic layer was dried over Na.sub.2SO.sub.4 and concentrated. The residue was purified by flash chromatography to give the intermediate alcohol.

(137) A suspension of NaH (0.83 mmol) in THF (2 mL) was cooled to 0 C., and then to the suspension was added the intermediate alcohol (0.17 mmol) in THF (4 mL) slowly. The mixture was refluxed for 2 h and then allowed to cool to room temperature. To the mixture was added A34 (0.11 mmol) in THF (2 mL) at that temperature dropwise. The reaction mixture was refluxed for 12 h and then allowed to cool to room temperature. After removal of the solvent by a rotary evaporator, the residue was dissolved in CHCl3 and filtered. The filtrate was evaporated by a rotary evaporator and chromatographed.

(138) To a solution of the PMB ether (0.1 mmol) in dichloroethane (5 mL), POCl.sub.3 (0.5 mmol) was added and stirred at room temperature. After completion of the reaction, it was quenched in ice water and the organic layer was separated and the aqueous layer was extracted with dichloroethane (25 mL). Combined organic layer was washed with brine solution, dried (Na.sub.2SO.sub.4), concentrated under reduced pressure and the residue was purified by column chromatography (silica gel, EtOAc:Hexane) to afford the corresponding alcohol A31.

Example 36.7

(139) ##STR00136##

(140) In the test tube were introduced epoxide A42 (5 mmol) and water (2 mL). Amine A41 (6 mmol) was added in one portion and the test tube was kept at 0 C. and warmed to room temperature under vigorous stirring for 24 h. Water (2 ml) was added and the aqueous mixture was extracted with 10 ml of ethyl acetate and dried over anhydrous Na.sub.2SO.sub.4, and solvent was removed under reduced pressure to give the intermediate -amino alcohol. The crude amine in EtOAc (2 mL) and sat. aq. NaHCO.sub.3 (1 mL) was treated with CbzCl (6 mmol) at room temperature and stirred for 5 h at that temperature. The mixture was extracted with EtOAc (35 mL), the organic fraction was dried over Na.sub.2SO.sub.4 and concentrated. The residue was purified by flash chromatography to give alcohol A40.

Example 36.8

(141) ##STR00137##

(142) Bromide A47 (8.26 mmol) was reacted with thiol A48 in MeOH (5 mL) adjusted to pH 9 with NaOMe. The mixture was stirred for 16 h at room temperature and concentrated under reduced pressure to give the intermediate PMB ether.

(143) To a solution of the PMB ether (0.1 mmol) in dichloroethane (5 mL), POCl.sub.3 (0.5 mmol) was added and stirred at room temperature. After completion of the reaction, it was quenched in ice water and the organic layer was separated and the aqueous layer was extracted with dichloroethane (25 mL). Combined organic layer was washed with brine solution, dried (Na.sub.2SO.sub.4), concentrated under reduced pressure and the residue was purified by column chromatography (silica gel, EtOAc:Hexane) to afford the corresponding alcohol A46.

Example 36.9

(144) ##STR00138##

(145) Acid A51 (1 mmol) and amine A50 (1 mmol) were coupled with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) hydrochloride (1 mmol) in 3.5 mL methanol for 3 h at room temperature. The mixture was diluted with EtOAc (20 mL) and extracted with water (10 mL), 1 M aq. HCl (10 mL) and sat. aq. NaHCO.sub.3 (10 mL). The organic layer was dried over Na.sub.2SO.sub.4 and concentrated. The residue was purified by flash chromatography to give alcohol A49.

Example 36.10

(146) ##STR00139##

(147) To a solution of A53 (80 mmol) in pyridine (60 mL) was added portionwise MsCl (80 mmol) with efficient stirring during 30 min at 20 C. After being stirred for 30 min, the reaction mixture was maintained for 20 h at room temperature, diluted with CH.sub.2Cl.sub.2 (100 mL), and washed with 2 N aq. HCl until the aqueous washings became acidic. The H.sub.2O layer was extracted with CH.sub.2Cl.sub.2 (350 mL). The combined organic extract was dried (Na.sub.2SO.sub.4), concentrated, and concentrated to give the intermediate mesylate. Sodium benzylsulfide (4.1 mmol) was added portionwise to a stirred solution of the intermediate mesylate (2.7 mmol) in DMF (5 mL) at room temperature. After 3 h, the mixture was diluted with toluene (100 mL), washed with water (25 mL) and brine (25 mL), dried (MgSO.sub.4), and concentrated in vacuo. The residue was purified by flash chromatography to give thioether A52.

Example 36.11

(148) ##STR00140##

(149) To a solution of the diol A55 (8 mmol) in pyridine (6 mL) was added portionwise TsCl (8 mmol) with efficient stirring during 30 min at 20 C. After being stirred for 30 min, the reaction mixture was maintained for 20 h at room temperature, diluted with CH.sub.2Cl.sub.2 (100 mL), and washed with 2 N aq. HCl until the aqueous washings became acidic. The H.sub.2O layer was extracted with CH.sub.2Cl.sub.2 (350 mL). The combined organic extract was dried (Na.sub.2SO.sub.4) and concentrated to give the intermediate tosylate.

(150) A solution of the intermediate tosylate (0.67 mmol) in 5 mL of dry HMPA was cooled in an ice bath under N.sub.2. This mixture was added to a cold solution of NaSBn (10 mmol) in 20 mL of dry HMPA (prepared from 400 mg of sodium and excess BnSH in dry ether, which was subsequently removed and replaced with HMPA). After the addition, the solution was stored in the freezer (15 C.) for 14 h. It was then treated with 100 mL of water and extracted three times with ether. The ether extracts were washed four times with water and dried over MgSO.sub.4. The solvent was then removed under vacuum and the residue (140 mg) chromatographed over silica gel to give thioether A54.

Example 36.12

(151) ##STR00141##

(152) To freshly distilled CH.sub.2Cl.sub.2 (20 mL) was added Et.sub.2Zn (1.0 M in hexanes) (20.0 mmol) under N.sub.2. The solution was cooled in an ice bath and a solution of trifluoroacetic acid (20.0 mmol) in CH.sub.2Cl.sub.2 (10 mL) was then dripped into the reaction mixture via syringe. Upon stirring for 20 min, a solution of CH.sub.2I.sub.2 (20.0 mmol) in CH.sub.2Cl.sub.2 (10 mL) was added. After an additional 20 min of stirring, a solution of diol A58 (10.0 mmol) in CH.sub.2Cl.sub.2 (10 mL) was added, and the ice bath was removed. After an additional 30 min of stirring, the reaction mixture was quenched with 0.1 N HCl (50 mL) (alternatively with saturated aqueous NH.sub.4Cl or Et.sub.3N followed by saturated aqueous NaHCO.sub.3) and hexanes (25 mL), and the layers were separated. The aqueous layer was extracted with hexanes. The combined, organic layers were washed with saturated NaHCO.sub.3, H.sub.2O, and brine and then dried (Na.sub.2SO.sub.4), filtered, concentrated, and purified by column chromatography (hexanes/ether=50/1) to give diol A57.

(153) To a solution of the diol A57 (8 mmol) in pyridine (6 mL) was added portionwise TsCl (8 mmol) with efficient stirring during 30 min at 20 C. After being stirred for 30 min, the reaction mixture was maintained for 20 h at room temperature, diluted with CH.sub.2Cl.sub.2 (100 mL), and washed with 2 N aq. HCl until the aqueous washings became acidic. The H.sub.2O layer was extracted with CH.sub.2Cl.sub.2 (350 mL). The combined organic extract was dried (Na.sub.2SO.sub.4) and concentrated to give the intermediate tosylate.

(154) A solution of the intermediate tosylate (0.67 mmol) in 5 mL of dry HMPA was cooled in an ice bath under N.sub.2. This mixture was added to a cold solution of NaSBn (10 mmol) in 20 mL of dry HMPA (prepared from 400 mg of sodium and excess BnSH in dry ether, which was subsequently removed and replaced with HMPA). After the addition, the solution was stored in the freezer (15 C.) for 14 h. It was then treated with 100 mL of water and extracted three times with ether. The ether extracts were washed four times with water and dried over MgSO.sub.4. The solvent was then removed under vacuum and the residue (140 mg) chromatographed over silica gel to give thioether A56.

Example 36.13

(155) ##STR00142##

(156) To a solution of diol A61 (16.1 mmol), triethylamine (24.1 mmol), and DMAP (0.16 mmol) in CH.sub.2Cl.sub.2 (120 mL) was added TBDMSCI (19.3 mmol) in 5 portions over 1 h at 0 C. The resulting heterogeneous reaction mixture was gradually warmed to rt. The mixture was stirred for 12 h before dilution with water and CH.sub.2Cl.sub.2. The organic layer was washed successively with solutions of saturated aq NaHCO.sub.3, saturated aq NH.sub.4Cl, water and brine, dried over MgSO.sub.4, filtered, and concentrated in vacuo. The pale yellow oil was purified by vacuum chromatography to give the intermediate silyl ether.

(157) A mixture of the intermediate silyl ether (1.0 mmol) and catalyst (Rh or Ru on activated carbon, N.E. Chemcat; 10 wt % of substrate) in iPrOH (1 mL) in a sealed tube was stirred at 60 C. at 5 atm H.sub.2. After cooling to room temperature, the reaction mixture was diluted with MeOH (20 mL) and the catalyst was removed by filtration through a membrane filter (Millipore, Millex-LH, 0.45 mm). The filtrate was concentrated in vacuo to give the corresponding decalin.

(158) To a solution of the intermediate decalin alcohol (8 mmol) in pyridine (6 mL) was added portionwise TsCl (8 mmol) with efficient stirring during 30 min at 20 C. After being stirred for 30 min, the reaction mixture was maintained for 20 h at room temperature, diluted with CH.sub.2Cl.sub.2 (100 mL), and washed with 2 N aq. HCl until the aqueous washings became acidic. The H.sub.2O layer was extracted with CH.sub.2Cl.sub.2 (350 mL). The combined organic extract was dried (Na.sub.2SO.sub.4) and concentrated to give tosylate A60.

(159) A solution of tosylate A60 (0.67 mmol) in 5 mL of dry HMPA was cooled in an ice bath under N.sub.2. This mixture was added to a cold solution of NaSBn (10 mmol) in 20 mL of dry HMPA (prepared from 400 mg of sodium and excess BnSH in dry ether, which was subsequently removed and replaced with HMPA). After the addition, the solution was stored in the freezer (15 C.) for 14 h. It was then treated with 100 mL of water and extracted three times with ether. The ether extracts were washed four times with water and dried over MgSO.sub.4. The solvent was then removed under vacuum and the residue (140 mg) chromatographed over silica gel to give the intermediate silyl ether.

(160) The intermediate silyl ether (0.235 mmol) was dissolved in THF (1 mL) at room temperature, followed by addition of 70% HF.pyridine (0.2 mL). After stirring for two days, the reaction mixture was carefully quenched with sat. aq. NaHCO.sub.3 and the resulting solution was diluted with EtOAc. The organic layer was washed with brine, dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography to give alcohol A59.

Example 36.14

(161) ##STR00143##

(162) A solution of squaric ethyl ester A67 (0.5 mmol), A68 (100 mg, 0.588 mmol) and Et.sub.3N (15 drops) in CH.sub.2Cl.sub.2 (10 mL) was stirred at room temperature overnight. Then it was concentrated under reduced pressure. The resulting crude residue, A66 (1.1 mmol) and Et.sub.3N (15 drops) in CH.sub.2Cl.sub.2 (10 mL) was stirred at room temperature overnight. Then it was concentrated under reduced pressure and purified by column chromatography. PMB-ether (0.444 mmol) was dissolved in acetone (4.5 ml) and water (0.5 ml). CAN (0.845 mmol) was added as a solid, followed by the dropwise addition of a solution of CAN (0.845 mmol) in acetone (0.9 ml) and water (0.1 ml) over 70 minutes. After a further 15 minutes, the reaction was poured into aqueous sodium bicarbonate solution, and extracted with chloroform. The product was purified on chromatography to give 65.

Example 36.15

(163) ##STR00144##

(164) Triethylamine (0.359 mL) was added to a solution of commercially available S-benzyl-(R)-cysteinol (2.56 mmol) in THF (7.50 mL). After 10 min of stirring, di-tert-butyldicarbonate (2.56 mmol) was added at 0 C. The reaction mixture was stirred for 2 h at room temperature. The solvent was then evaporated under reduced pressure; the residue was dissolved in ethyl acetate and washed with water. The organic layer was separated, dried with Na.sub.2SO.sub.4, filtered, and evaporated under reduced pressure to yield A69 as a colorless oil.

Example 37

Further Examples of Saccharides According to the Present Invention

(165) ##STR00145## ##STR00146##

Example 38

Synthesis of glycoconiugatesConjugation of 2-acetamido-4-amino-2,4,6-trideoxy--D-galactopyranosyl-(14)-(-D-galactopyranosyl)uronate-(13)-(-D-galactopyranosyl)uronate-(11)-2-(thio)ethanol to CRM197

(166) To a stirred solution of CRM197 (2 mg, 34.5 nmol) in 0.1 M sodium phosphate buffer (NaPi) pH 7.4 (1.33 mL) was added at room temperature a solution of N-Succinimidyl-3-(bromoacetamido)propionate (SBAP) (1.05 mg, 3.4 mol) in DMF (40 L). The mixture was stirred for 1 h at that temperature, and concentrated using membrane filtration (Amicon 4 mL Ultra centrifuge membranes, 10 kDa cut-off). The protein solution was diluted to 4 mL with sterile water and concentrated again. This process was repeated three times and the solution was diluted to 0.5 mL using sterile water. 20 L were taken for analysis, and the protein solution was re-buffered to 0.1 M NaPi pH 8.0 (0.5 mL) using membrane filtration. Disulfide 18* (1.44 mg, 2.33 mol resp. to the monomer) in 0.1 M NaPi pH 8.0 (0.2 mL) was treated at room temperature with tris(2-carboxyethyl)phosphine (TCEP, 25 L of a 100 mM stock solution with pH 7.4), left for 1 h at that temperature under an argon atmosphere and added to the solution of the activated protein. The mixture was stirred at room temperature for 16 h, and washed with sterile water using membrane filtration (see above). Another analytical sample was taken, and the solution was re-buffered to 0.1 M NaPi pH 7.4 (0.5 mL). The glycoconjugate was then treated at room temperature with L-cysteine (0.625 mg, 5.1 mol) in 100 l sterile water. The mixture was left for 2 h at that temperature and purified by membrane filtration. Incorporation of glycan into the glycoconjugate was assessed by MALDI-TOF-MS (positive mode):

(167) Molecular Weight Measured:

(168) CRM197: 58100 m/z

(169) CRM197-SBAP conjugate: 61700 m/z (incorporation of approximatively 19 SBAP groups)

(170) CRM197-SBAP-glycoconjugate: 66000 m/z (incorporation of approximatively 5.9 molecules of 2-acetamido-4-amino-2,4,6-trideoxy--D-galactopyranosyl-(1.fwdarw.4)-(-D-galactopyranosyl)uronate-(1.fwdarw.3)-(-D-galactopyranosyl)uronate-(1.fwdarw.1)-2-(thio)ethanol).

Example 39

Immunization Experiment

(171) Mice (6-8 week old female NMRI mice, Charles River) were immunized s. c. with CRM197-SBAP-glycoconjugate synthesized in Example 38 (corresponding to 4 g synthetic glycan) formulated with or without Alum (Alhydrogel, Brenntag) at a total volume of 100 L at days 0, 14 and 28. Control groups comprised mice treated equally with Alum only or PBS. Blood was collected at days 0, 14, 28 and 35 and the immune response was assessed by glycan microarray and ELISA.

(172) An immune response against the native Sp1 polysaccharide was found in a subset of mice immunized with glycoconjugate adjuvanted with Alum with an endpoint titer of 500 and 2500 at day 35 compared to day 0, respectively.