Oligosaccharide conjugates and methods of use

Abstract

There is provided a method of detecting in a sample the presence of an anti-M and/or anti-A and/or anti-C/Y antibody, the method comprising contacting the sample with a diagnostic conjugate provided according to the invention, comprising an oligosaccharide which comprises at least two units of 4,6-dideoxy-4-acylamido-α-pyranose and comprising at least one -(1-3)-link between adjacent 4,6-dideoxy-4-acylamido-α-pyranose units, in which the carbon at position 5 in the pyranose is linked to an R group, where R is independently selected from —CH.sub.2OH, —H or an alkyl group having at least one C atom, the oligosaccharide being covalently linked to a non-saccharide molecule or to a surface.

Claims

1. A method of detecting in a sample the presence of an anti-M and/or anti-A and/or anti-C/Y antibody, the method comprising contacting the sample with a diagnostic conjugate comprising an oligosaccharide which consists of 2-15 units of 4,6-dideoxy-4-acylamido-α-pyranose, wherein at least two 4,6-dideoxy-4-acylamido-α-pyranose units are linked with a -(1-3)- link, in which the carbon at position 5 in the pyranose is linked to an R group, where R is independently selected from —CH.sub.2OH, —H or an alkyl group having at least one C atom, the oligosaccharide being covalently linked to a non-saccharide molecule or to a solid entity, wherein detection of binding of an antibody in the sample to the diagnostic conjugate is indicative of the presence in the sample of an anti-M and/or anti-A and/or anti-C/Y antibody.

2. The method of claim 1, wherein the diagnostic conjugate is a universal antigen, capable of binding to an anti-M antibody or to an anti-A antibody or to an anti-C/Y antibody.

3. The method of claim 1, wherein the oligosaccharide comprises no does not have more than one -(1-3)- link.

4. The method of claim 1, wherein the oligosaccharide consists of two, three or four 4,6-dideoxy-4-acylamido-α-pyranose units.

5. The method of claim 4, wherein the diagnostic conjugate is a specific M-antigen, capable of preferentially binding to an anti-M antibody and wherein detection of binding of an antibody in the sample to the diagnostic conjugate is indicative of the presence in the sample of an anti-M antibody.

6. The method of claim 1, wherein the oligosaccharide is a tetrasaccharide of Formula VII, wherein Formula VII is 4,6-dideoxy-4-acylamido-α-pyranosyl-(1-2)-4,6-dideoxy-4-acylamido-α-pyranosyl-(1-3)-4,6-dideoxy-4-acylamido-α-pyranosyl-(1-2)-4,6-dideoxy-4-acylamido-α-pyranose.

7. The method of claim 1, wherein the diagnostic conjugate comprises an oligosaccharide which consists of 6-15 4,6-dideoxy-4-acylamido-α-pyranose units and comprising overlapping tetrasaccharides of Formula VII, such that a third and fourth 4,6-dideoxy-4-acylamido-α-pyranose units in one tetrasaccharide form a first and second 4,6-dideoxy-4-acylamido-α-pyranose units in the next tetrasaccharide, to form an oligosaccharide in which the links between contiguous 4,6-dideoxy-4-acylamido-α-pyranose units are alternating -(1-2)- and -(1-3)- links; in which the carbon at position 5 in the pyranose is linked to an R group, where R is independently selected from —CH.sub.2OH, —H or an alkyl group having at least one C atom.

8. The method of claim 1, wherein the diagnostic conjugate comprises oligosaccharide which consists of 7-15 4,6-dideoxy-4-acylamido-α-pyranose units and comprising overlapping tetrasaccharides of Formula VII, such that a fourth 4,6-dideoxy-4-acylamido-α-pyranose unit in one tetrasaccharide forms the a 4,6-dideoxy-4-acylamido-α-pyranose unit in the next tetrasaccharide; in which the carbon at position 5 in the pyranose is linked to an R group, where R is independently selected from —CH.sub.2OH, —H or an alkyl group having at least one C atom.

9. The method of claim 1 wherein, in the oligosaccharide, R is methyl, ethyl or butyl.

10. The method of claim 1, wherein at least one 4,6-dideoxy-4-acylamido-α-pyranosyl unit is 4,6-dideoxy-4-formamido-α-D-mannopyranosyl.

11. The method of claim 1, wherein the non-saccharide molecule is selected from the group consisting of a protein, Bovine Serum Albumin (BSA), a non-protein carrier molecule comprising hydrophobic elements, and a fluorophore.

12. The method of claim 1, wherein the oligosaccharide is linked to a solid entity via a protein, via Bovine Serum Albumin (BSA), or via hydrazone conjugation.

13. The method of claim 1, wherein the sample is a biological sample obtained from an animal.

14. The method of claim 13, wherein the biological sample is a blood, plasma, serum, tissue, saliva or milk sample.

15. A method of detecting in a sample the presence of an anti-Brucella antibody, the method comprising contacting the sample with a diagnostic conjugate comprising an oligosaccharide which consists of two, three or four units of 4,6-dideoxy-4-acylamido-α-pyranose, wherein two 4,6-dideoxy-4-acylamido-α-pyranose units are linked with a -(1-3)- link, in which the carbon at position 5 in the pyranose is linked to an R group, where R is independently selected from —CH.sub.2OH, —H or an alkyl group having at least one C atom, the oligosaccharide being covalently linked to a non-saccharide molecule or to a solid entity, wherein detection of binding of an antibody in the sample to the diagnostic conjugate is indicative of the presence in the sample of an anti-Brucella antibody, and wherein the anti-Brucella antibody is not an anti-B. suis biovar 2 or an anti-B. inopinata BO2 antibody.

16. A method of determining that an animal is or has been infected with a Brucella organism, the method comprising contacting a first sample obtained from the animal with a first diagnostic conjugate and detecting binding of the first diagnostic conjugate to at least one antibody present in the sample, the first diagnostic conjugate comprising an oligosaccharide which consists of 6-15 units of 4,6-dideoxy-4-acylamido-α-pyranose, wherein at least two 4,6-dideoxy-4-acylamido-α-pyranose units are linked with a -(1-3)- link, in which the carbon at position 5 in the pyranose is linked to an R group, where R is independently selected from —CH.sub.2OH, —H or an alkyl group having at least one C atom, the oligosaccharide being covalently linked to a non-saccharide molecule or to a solid entity; the method further comprising a second step of contacting a second sample obtained from the animal with a second diagnostic conjugate comprising an oligosaccharide which comprises two, three or four units of 4,6-dideoxy-4-acylamido-α-pyranose, wherein at least two 4,6-dideoxy-4-acylamido-α-pyranose units are linked with a -(1-3)- link, in which the carbon at position 5 in the pyranose is linked to an R group, where R is independently selected from —CH.sub.2OH, —H or an alkyl group having at least one C atom, the oligosaccharide being covalently linked to a non-saccharide molecule or to a solid entity; and detecting binding of the second diagnostic conjugate to at least one antibody present in the sample; wherein detection of binding in both the first and the second steps is indicative of infection of the animal by a Brucella organism, and wherein the Brucella organism is not B. suis biovar 2 or B. inopinata BO2.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) Embodiments of the invention will now be described, by way of example only, with reference to FIGS. 1-27 in which:

(2) FIG. 1 shows target pentasaccharide (1) which exhibits preferred binding to M-specific antibodies and nonasaccharide (2) designed to bind both A- and M-specific antibodies;

(3) FIG. 2 shows results (expressed as a percentage of a common positive control) from the B. melitensis 16M OPS iELISA (x-axis) and Y. enterocolitica O:9 OPS iELISA (y-axis); the solid diamonds represent the results for sera from cattle confirmed as infected with B. abortus biovar 1 (n=45) and the crosses represent the results for sera from non-Brucella infected cattle that are positive in one or more conventional serodiagnostic assays (n=68);

(4) FIG. 3A shows results (expressed as a percentage of a common positive control) for B. melitensis 16M OPS iELISA (x-axis) and Y. enterocolitica O:9 OPS iELISA (y-axis) ELISAs; the solid diamonds represent the results for sera from swine confirmed as infected with B. suis biovar 1 (n=41) and the crosses represent the results for sera from non-Brucella infected swine from herds with one or more animals that are positive in one or more conventional serodiagnostic assays (n=52);

(5) FIG. 3B shows results (expressed as a percentage of a common positive control) for B. abortus S99 OPS iELISA (x-axis) and Y. enterocolitica O:9 OPS iELISA (y-axis) ELISAs; the solid diamonds represent the results for sera from swine confirmed as infected with B. suis biovar 1 (n=41) and the crosses represent the results for sera from non-Brucella infected swine from herds with one or more animals that are positive in one or more conventional serodiagnostic assays (n=52);

(6) FIG. 4 shows selected ion count for the tetrasaccharide (m/z=711.3) (A) from B. melitensis 16M core OPS (three significant peaks visible at 8:05, 8.50 & 11:10 mins:secs), (B) from B. abortus S99 core OPS (five significant peaks visible at 8:05, 8:50, 9:20, 10:05 and 10:40 mins:secs), (C) from Y. enterocolitica O:9 core OPS (six significant peaks visible at 8:05, 9:20, 9:55, 10:10, 10:40 and 11:30 mins:secs); (D) from B. abortus S99 core OPS eluted from an affinity chromatography column conjugated with anti-Brucella mAb BM40 (three significant peaks visible at 8:50, 10:30 and 11:15 mins:secs); all core OPS hydrolysed and purified (by size exclusion chromatography) and analysed on a graphitised carbon HPLC column online with ESI-triple quadrupole mass spectrometer;

(7) FIG. 5 shows inhibition of BM40 mAb binding to solid phase B. melitensis 16M OPS antigen (y-axis) by varied concentrations of competing liquid phase antigen (x-axis), with three types of competing OPS antigens (as shown in legend) evaluated in addition to the TSM antigen according to the invention;

(8) FIG. 6 shows inhibition of rabbit anti-′M′ monospecific antisera binding to solid phase B. melitensis 16M core-OPS antigen (y-axis) (FIG. 7A) or inhibition of rabbit anti-‘A’ monospecific antisera binding to solid phase B. abortus S99 core-OPS antigen (y-axis) (FIG. 7B) by varied concentrations of competing liquid phase antigen (x-axis), with three types of competing OPS antigens evaluated (as shown in legend) in addition to the TSM (tetrasaccharide) antigen according to the invention;

(9) FIG. 7 shows the results (expressed as a percentage of a common positive control) from the B. melitensis 16M OPS iELISA (x-axis) and TSM antigen iELISA (y-axis); the solid diamonds represent the results for sera from cattle confirmed as infected with B. abortus biovar 1 (n=45) and the crosses represent the results for sera from non-Brucella infected cattle that are positive in one or more conventional serodiagnostic assays (n=68);

(10) FIG. 8 shows ELISA titration curves for Brucella A- and M-specific mAbs, YsT9-1 and Bm10; pentasaccharide conjugate 42 (right panel) and nona-saccharide conjugate 43 (left panel);

(11) FIG. 9 shows results (expressed as a percentage of a common positive control) from the BSA-nonasaccharide conjugate iELISA (x-axis) and BSA-pentasaccharide conjugate iELISA (y-axis); the solid diamonds represent the results for sera from cattle confirmed as infected with B. abortus biovar 1 (n=45) and the crosses represent the results for sera from non-Brucella infected cattle that are positive in one or more conventional serodiagnostic assays (n=68);

(12) FIG. 10 shows results (expressed as a percentage of a common positive control) from the BSA-tetrasaccharide conjugate iELISA (x-axis) and BSA-disaccharide conjugate iELISA (y-axis); the solid diamonds represent the results for sera from cattle confirmed as infected with B. abortus biovar 1 (n=45) and the crosses represent the results for sera from non-Brucella infected cattle that are positive in one or more conventional serodiagnostic assays (n=68);

(13) FIG. 11 shows results (expressed as a percentage of a common positive control) from the BSA-trisaccharide conjugate (with terminal α-1,3 link) iELISA (x-axis) and BSA-trisaccharide conjugate (with terminal α-1,2 link) iELISA (y-axis); the solid diamonds represent the results for sera from cattle confirmed as infected with B. abortus biovar 1 (n=45) and the crosses represent the results for sera from non-Brucella infected cattle that are positive in one or more conventional serodiagnostic assays (n=68);

(14) FIG. 12 shows ROC Curves generated from the results of the Y. enterocolitica O:9 OPS, B. melitensis 16M OPS, BSA-pentasaccharide, BSA-tetrasaccharide and BSA-disaccharide conjugate iELISAs as applied to sera from sera from cattle confirmed as infected with B. abortus biovar 1 (n=45) and sera from non-Brucella infected cattle that are positive in one or more conventional serodiagnostic assays (n=68);

(15) FIG. 13 shows the results from the B. melitensis 16M OPS iELISA as applied to sera from four cattle experimentally infected with Brucella abortus strain 544 (Brucella #1 to #4, solid lines) and four cattle experimentally infected with Y. enterocolitica O:9 (YeO:9 #1 to #4, dashed lines); samples were taken on weeks 3, 7, 16 and 24 post infection;

(16) FIG. 14 shows the results from the BSA-nonasaccharide conjugate iELISA as applied to sera from four cattle experimentally infected with Brucella abortus strain 544 (Brucella #1 to #4, solid lines) and four cattle experimentally infected with Y. enterocolitica O:9 (YeO:9 #1 to #4, dashed lines); samples were collected, on weeks 3, 7, 16 and 24 post infection;

(17) FIG. 15 shows the results from the BSA-pentasaccharide conjugate iELISA as applied to sera from four cattle experimentally infected with Brucella abortus strain 544 (Brucella #1 to #4, solid lines) and four cattle experimentally infected with Y. enterocolitica O:9 (YeO:9 #1 to #4, dashed lines); samples were taken on weeks 3, 7, 16 and 24 post infection;

(18) FIG. 16 shows the results from the B. melitensis 16M OPS iELISA (x-axis) and the BSA-pentasaccharide conjugate iELISA (y-axis) as applied to sera (n=16) from four cattle experimentally infected with Brucella abortus strain 544 (solid diamonds), and sera (n=16) from four cattle experimentally infected with Y. enterocolitica O:9 (crosses). Samples were taken on weeks 3, 7, 16 and 24 post infection; the scatter plot only distinguishes between samples from different infection types, not by animal and time post infection;

(19) FIG. 17 shows the results from the BSA-tetrasaccharide conjugate iELISA as applied to sera from four cattle experimentally infected with Brucella abortus strain 544 (Brucella #1 to #4, solid lines) and four cattle experimentally infected with Y. enterocolitica O:9 (YeO:9 #1 to #4, dashed lines); samples were taken on weeks 3, 7, 16 and 24 post infection;

(20) FIG. 18 shows the results from the BSA-disaccharide conjugate iELISA as applied to sera from four cattle experimentally infected with Brucella abortus strain 544 (Brucella #1 to #4, solid lines) and four cattle experimentally infected with Y. enterocolitica O:9 (YeO:9 #1 to #4, dashed lines); samples were taken on weeks 3, 7, 16 and 24 post infection;

(21) FIG. 19 shows the results from the BSA-tetrasaccharide conjugate iELISA (x-axis) and the BSA-disaccharide conjugate iELISA (y-axis) as applied to sera (n=16) from four cattle experimentally infected with Brucella abortus strain 544 (solid diamonds), and sera (n=16) from four cattle experimentally infected with Y. enterocolitica O:9 (crosses). Samples were taken on weeks 3, 7, 16 and 24 post infection; the scatter plot only distinguishes between samples from different infection types, not by animal and time post infection;

(22) FIG. 20 shows results (expressed as a percentage of a common positive control) from the BSA-nonasaccharide conjugate iELISA (x-axis) and BSA-pentasaccharide conjugate iELISA (y-axis); the solid diamonds represent the results for sera from cattle confirmed as infected with B. abortus biovar 1 (n=45) and the crosses represent the results for sera from randomly sampled brucellosis free cattle (n=125);

(23) FIG. 21 shows results (expressed as a percentage of a common positive control) from the BSA-tetraasaccharide conjugate iELISA (x-axis) and BSA-disaccharide conjugate iELISA (y-axis); the solid diamonds represent the results for sera from cattle confirmed as infected with B. abortus biovar 1 (n=45) and the crosses represent the results for sera from randomly sampled brucellosis free cattle (n=125);

(24) FIG. 22 shows the results (expressed as a percentage of a common positive control) for B. melitensis 16M OPS iELISA (x-axis) and TSM antigen (y-axis) ELISAs; the solid diamonds represent the results for sera from sheep and goats infected with B. melitensis biovar 3 (n=61) and the open triangles represent the results for sera from non-Brucella infected sheep and goats that have been randomly sampled within Great Britain (n=94);

(25) FIG. 23 shows the results (expressed as a percentage of a common positive control) for the BSA-pentasaccharide conjugate iELISA (x-axis) and BSA-nonasaccharide conjugate (y-axis) ELISAs; the solid diamonds represent the results for sera from sheep and goats infected with B. melitensis biovar 3 (n=61) and the open triangles represent the results for sera from non-Brucella infected sheep and goats that have been randomly sampled within Great Britain (n=94);

(26) FIG. 24 shows titration of human serum (#2) from a human patient diagnosed Brucella suis positive by bacterial culture, against copovidone-conjugated disaccharide conjugates 99a and 99b;

(27) FIG. 25 shows titration of monoclonal antibodies and human serum #2 against ELISA plates coated with low loading, “Brucella A type” hexasaccharide conjugate 100b;

(28) FIG. 26 shows titration of monoclonal antibodies and human serum #2 against ELISA plates coated with universal antigen “Brucella A and M type” hexasaccharide conjugate 98; and

(29) FIG. 27 shows the binding of HRP conjugated BM40 anti-M mAb, expressed as iELISA optical density (OD) on the y-axis, against three BSA-oligosaccharide conjugates shown as three separate lines: the disaccharide, a trisaccharide and the tetrasaccharide

EXAMPLES

(30) Oligosaccharide and Conjugate Synthesis

(31) General methods are provided in the section below headed “General Synthesis Methods”.

(32) Pentasaccharide 1 (FIG. 1; e.g. Formula XI) was selected as the largest sized antigen that might selectively exhibit M-type characteristics with limited cross reaction with A-specific antibodies. Nonasaccharide 2 (Formula XV above) was considered to be an antigen that would contain two A- and one M-type epitopes, which would serve as a universal antigen to detect antibodies in animals or humans infected by B. abortus, B. melitensis and B. suis.

(33) The large size of the target oligosaccharides 1 and 2, the incorporation of an internal 1,3 linkage and a tether for antigen synthesis was not previously attempted (Peters & Bundle (1989) Can. J. Chem. 67, 497-502) and necessitated the development of an improved synthetic strategy. The linker 5-methoxycarbonylpentanol (Lemieux et al. (1975) J. Am. Chem. Soc. 97, 4076-4083; Ogawa et al. (1996) Carbohydr. Res. 293, 173-94) was chosen for its compatibility with the strategy employed to deprotect the assembled oligosaccharides and for its flexibility in offering several routes for subsequent conjugation to protein to create glycoconjugate antigens (Kamath et al. (1996) Glycoconjugate 13, 315-319). Two distinct strategies were employed to synthesis first the penta- and nonasaccharides, with a second revised strategy to synthesise di-, tri-, tetra- and hexasaccharides. The second strategy employed related, but slightly modified, protection schemes for the construction of the monosaccharide and disaccharide synthons.

(34) Synthetic Strategy 1

(35) Well-established methods were utilised to synthesize compounds 3-6, as outlined below (Bundle et al. (1998) Carbohydr. Res. 174, 239-251). Lewis acid catalyzed glycosidation of 6 afforded the allyl glycoside 7, which was transesterified to 8. Formation of a 2,3-orthoester derivative which undergoes regioselective opening afforded the selectively protected building block 9 (Scheme 1).

(36) ##STR00003##

(37) We used N-phenyl trifluoroacetimidates for glycosylation reactions since this donor has been shown to be more efficient than the corresponding trichloroacetimidate derivative for glycosylations involving 6-deoxy sugar donors (Hanashima et al. (2007) Org. Lett. 9, 1777-1779). Selective deacetylation of 5 gave 10, and then reaction with the N-phenyl trifluoroacetimidoyl chloride in presence of cesium carbonate as base gave the glycosyl N-phenyl trifluoroacetimidate donor 11 (Hanashima et al. (2007) Org. Lett. 9, 1777-1779). Monosaccharide 5 was also converted to thioglycoside donor 12 and transesterification afforded the acceptor 13 (Peters & Bundle (1989) Can. J. Chem. 67, 491-496). Glycosylation of 13 by 11 was performed in the presence of trimethylsilyl trifluoromethanesulfonate to give disaccharide 14 in 94% yield with complete stereocontrol and without detectable amounts of β-anomer (Scheme 2).

(38) ##STR00004##

(39) The 1,3-linked trisaccharide building block 15 was created as its allyl glycoside since the selective removal of this anomeric protecting group allows facile access to a hemiacetal and subsequently an imidate leaving group (Du et al. (2001) Tetrahedron 57, 1757-1763). Glycosylation reactions were tried with allyl as a leaving group but all attempts to do so failed (Wang et al. (2007) J. Org. Chem. 72, 5870-2873). Consequently, 15 was selectively deprotected with palladium chloride in acetic acid (Du et al. (2001) Tetrahedron 57, 1757-1763) to give hemiacetal 16 which was in turn converted to the N-phenyl trifluoroacetimidate donor 17 (Scheme 3).

(40) ##STR00005##

(41) Glycosylation of 5-methoxycarbonylpentanol by thioglycoside 14 gave moderate to poor yields due to the low reactivity of acceptors of this type (Lemieux et al. (1975) J. Am. Chem. Soc. 97, 4076-4083). Hydrolysis of the thioethyl glycoside 14 gave hemiacetal 18 which was converted it to imidate 19. The six carbon linker 20 (El Fangour et al. (2004) J. Org. Chem. 69, 2498-1503) which was glycosylated by 19 to give the protected disaccharide glycoside 21 (Hou & Ková{hacek over (c)} (2010) Carbohydr. Res. 345, 999-1007). Transesterification of 21 gave the tether glycoside acceptor 22 (Scheme 4).

(42) ##STR00006##

(43) Pentasaccharide 23 was obtained from building blocks trisaccharide 17 and disaccharide glycoside 22 in 68% yield using TMSOTf as the activator (Scheme 5). Stepwise deprotection followed the sequence: deacetylation to give 24 in quantitative yield, azido group reduction with hydrogen sulfide to give 25. Compound 25 was directly formylated by a mixed anhydride (acetic anhydride/formic acid 2:1) to give 26 (Bundle et al. (1988) Carbohydr. Res. 174, 239-251). Following introduction of the N-formamido groups, NMR analyses of all subsequent compounds became difficult due to the presence of E/Z rotamers for each formyl group, leading to a potential mixture of 32 isomers. Their identity was confirmed by a limited set of characteristic NMR resonances and high resolution mass measurements. Pentasaccharide 1 was obtained by hydrogenolysis of benzyl ethers.

(44) ##STR00007##

(45) The synthesis of nonasaccharide 2 was envisaged as the creation of a pentasaccharide donor terminated by a 1,3 linkage which would then allow for a pentasaccharide donor with a participating group at C-2 to guide the stereoselective α-glycosylation of an exclusively 1,2-linked tetrasaccharide. To achieve the synthesis of the pentasaccharide donor, compound 14 was deprotected to give the corresponding acceptor 27 which was glycosylated by imidate donor 19. Tetrasaccharide 28 was formed in toluene at 100° C. as described for 21 (Hou & Ková{hacek over (c)} (2010) Carbohydr. Res. 345, 999-1007). Tetrasaccharide thioglycoside 28 was used directly as the donor for glycosylation of the monosaccharide glycoside 9 to give the α1,3-linkage. The allyl group of pentasaccharide 29 was then removed (Du et al. (2001) Tetrahedron 57, 1757-1763) to give 30 and the imidate donor 31 was obtained following reaction with N-phenyl trifluoroacetimidoyl chloride (Scheme 6).

(46) ##STR00008##

(47) Tetrasaccharide tether glycoside 32 was obtained by a 2+2 glycosylation of disaccharide acceptor 22 by the disaccharide donor 19 employing the same condition used to prepare 28. Transesterification of 32 gave the tetrasaccharide acceptor 33 which was glycosylated by pentasaccharide donor 31 to give nonasaccharide 34 in 30% yield. The sequence of deprotection steps (deacetylation to 35, reduction of azide to 36, N-formylation to 37 and hydrogenation) to give 2 followed the order used to obtain pentasaccharide 1 (Scheme 7).

(48) ##STR00009##

(49) The final compounds 1 and 2 were purified by reverse phase HPLC. Full NMR assignments were performed on the azido penta and nonasaccharide derivatives 24 and 35. Selected characteristic NMR resonances and high resolution mass confirmed the identity of derivatives 26 and 37 and the target oligosaccharides 1 and 2.

(50) To enable conjugation to protein, pentasaccharide and nonasaccharide glycosides 1 and 2 were first converted to the respective amides 38 and 39 by reaction with ethylenediamine (Scheme 8). Reaction of 38 and 39 with dibutyl squarate gave the squarate half esters 40 and 41 which were isolated by reverse phase HPLC. The corresponding pentasaccharide and nonasaccharide bovine serum albumin (BSA) glycoconjugates 42 and 43 were prepared by reaction of a twenty to one molar ratio of 40 and 41 with BSA in borate buffer for 3 days. MALDI-TOF mass spectrometry indicated that each conjugate contained approximately 16 copies of the oligosaccharides per molecule of BSA.

(51) ##STR00010##
Synthetic Strategy 2

(52) This strategy set out to arrive at shorter oligosaccharides 44-47 that would provide M-specific antigens and achieve the level of discrimination described above.

(53) ##STR00011##

(54) Two additional compounds were synthesized; hexasaccharide 48 to provide a pure A epitope and compound 49 which provides an oligosaccharide of minimal size that encompasses both A and M epitopes. Oligosaccharide 49, along with the nonasaccharide 2, is a universal Brucella antigen. It corresponds to the terminal hexasaccharide of the Brucella O-antigen disclosed by Kubler-Kielb and Vinogradov (Carbohydr. Res. (2013) 378, 144-147).

(55) ##STR00012##

(56) In contrast to the synthesis of 1 and 2 where acetate esters and benzyl ethers were used, the synthesis of oligosaccharides 44-49 made use of benzoate esters and benzyl ether protecting groups to allow for the construction of 1,2 and 1,3 linkages. This distinct difference in protecting group strategy from the earlier synthesis of 1 and 2 allowed the use of trichloroacetimidate donors rather than the more difficult to prepare N-phenyl trifluoroacetimidates 11, 19 and 31. The synthons used to make oligosaccharides 44-49 were compounds 50-66. The monosaccharide imidates 53, 58 and 62 were synthesized as shown (Schemes 9 and 10). Two disaccharide thioglycosides 63 and 64 were prepared by glycosylation of 13 by imidates 53 and 62 (Scheme 10). The two 5-methoxycarbonylpentanol glycosides 65 and 66 were prepared by literature methods (Saksena et al. (2008) Carbohydr. Res. 343, 1693-1706; Saksena et al. (2005) Tetrahedron: Asymmetry 16, 187-197). Imidate 62 was used in the synthesis of 48 and 49.

(57) ##STR00013##

(58) The 1,3 linked disaccharide 67 was obtained by reaction of the imidate 53 with the acceptor 65. Trisaccharide 68 was obtained by reaction of the disaccharide thioglycoside 63 with the monosaccharide glycoside 65. Reaction of the monosaccharide imidate 58 with 66 gave the 1,2 linked disaccharide 69. When 69 was de-O-benzoylated the resulting disaccharide 70 could be glycosylated by the monosaccharide imidate 53 to give the trisaccharide 71 with a terminal 1,3 linkage. Disaccharide 70 also provides access to tetrasaccharide 72 when it was reacted with disaccharide thioglycoside 63 (Scheme 11).

(59) ##STR00014##

(60) Hexasaccharides 48 and 49 were elaborated on the monosaccharide glycoside 66 by glycosylation with the disaccharide thioglycoside 64 to give the trisaccharide 73 which after transesterification provides the alcohol acceptor 74, which serves as the common trisaccharide intermediate leading to both hexsaccharides (Scheme 12). The exclusively 1,2-linked hexasaccharide 48 was prepared by glycosylation of 74 by the disaccharide thioglycoside 64 to give pentasaccharide 75. Transesterification of the terminal benzoate group gave the pentasaccharide alcohol 76, which afforded hexasaccharide 77 after glycosylation by the imidate 53. Glycosylation of trisaccharide alcohol 74 by imidate 58 afforded tetrasaccharide 78. After transesterication of 78 the tetrasaccharide alcohol 79 is set up for introduction of a 1,3-linkage. Reaction with the disaccharide thioglycoside 63 gives the protected hexasaccharide 80.

(61) ##STR00015## ##STR00016##

(62) ##STR00017## ##STR00018##

(63) Deprotection of the six oligosaccharides 67, 68, 71, 72, 77 and 80 employed identical reaction conditions (Scheme 13). This involved transesterification to remove benzoate esters, reduction of the azide groups to amines, acylation of amines by formic anhydride to afford formamides and lastly catalytic hydrogenation to afford the target oligosaccharides as 5 methoxycarbonylpentanol glycosides 44-49.

(64) Conversion of the 5 methoxycarbonylpentanol glycosides 44-49 to antigens for diagnostic or vaccine applications followed a similar protocol to that described for the penta and nonasaccharides (Scheme 14).

(65) ##STR00019##

(66) A non-protein polymer aminated co-povidone was also used as an alternative and potential superior antigen for immunoassays. Representative conjugations are described for the disaccharide 44 and the hexasaccharide 48 (Scheme 15).

(67) ##STR00020##

(68) Potential vaccine candidates were synthesized by conjugating the two hexasaccharides 48 and 49 to monomeric tetanus toxoid. The squarate half esters 91 and 92 were each conjugated to tetanus toxoid to provide antigens 101 and 102 (Scheme 16).

(69) ##STR00021##
General Synthesis Methods

(70) Analytical TLC was performed on Silica Gel 60-F.sub.254 (Merck, Darmstadt) with detection by quenching of fluorescence and/or by charring with 5% sulfuric acid in ethanol. All commercial reagents were used as supplied. Column chromatography was performed on Silica Gel 230-400 mesh, 60 Å (Silicycle, Ontario) with HPLC quality solvents. Molecular sieves were crushed and stored in an oven at 150° C. and flamed dried under vacuum before use. Organic solutions were dried with anhydrous MgSO.sub.4 prior to concentration under vacuum at <40° C. (bath). All final compounds were purified by reverse phase chromatography performed on a Waters 600 HPLC system, using a Beckmann semi-preparative C-18 column (10×250 mm, 5μ) with a combination of acetonitrile and water as eluents. Products were detected with a Waters 2487 UV detector.

(71) Optical rotations were measured with a Perkin-Elmer 241 polarimeter for samples in a 10 cm cell at 21±2° C. [α].sub.D values are given in units of 10.sup.−1 deg cm.sup.2 g.sup.−1, with [α].sub.D.sup.20 indicating that the temperature was 20° C. and [α].sub.D.sup.21 indicating that the temperature was 21° C.

(72) .sup.1H NMR spectra were recorded on 500, 600 or 700 MHz spectrometers. First order proton chemical shifts δ.sub.H are referenced to either residual CHCl.sub.3 (δ.sub.H 7.27, CDCl.sub.3) or CD.sub.2HOD (δ.sub.H 3.30, CD.sub.3OD), or internal acetone (δ.sub.H 2.225, D.sub.2O). .sup.13C NMR spectra were recorded at 125 MHz, and chemical shifts are referenced to internal CDCl3 (δ 77.23) or external acetone (δ 31.07). The assignment of resonances for all compounds was made by two-dimensional homonuclear and heteronuclear chemical shift correlation experiments. Mass analysis was performed by positive-mode electrospray ionization on a hybrid sector-TOF mass spectrometer and for protein glycoconjugates by MALDI mass analysis, employing sinapinic acid as matrix.

(73) The numbering used for compounds 4-41 is as follows:

(74) ##STR00022##
and for compounds 42-92:

(75) ##STR00023##

Methyl 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (4)

(76) A solution of 3 (Bundle et al. (1988) Carbohydr. Res. 174, 239-251) (1.09 g, 5.36 mmol) and Bu.sub.2SnO (1.5 g, 6 mmol) in toluene (50 mL) was stirred at 140° C. for 2 h. Then, after cooling down, benzyl bromide (0.7 mL, 5.9 mmol) and Bu.sub.4NBr (1.9 g, 5.9 mmol) were added and the mixture was stirred overnight at 65° C. After evaporation of the solvent, a purification on a silica gel column (hexane/ethyl acetate 8:1) gave pure 4 (1.38 g, 87%). The NMR parameters are in agreement with the literature (Boschiroli et al. (2001) Curr. Op. Microbiol. 4, 58-64): .sup.1H NMR (500 MHz, CDCl.sub.3): δ 7.5-7.3 (m, 5H; H—Ar), 4.72 (d, .sup.3J.sub.1,2=1.7 Hz, 1H; H-1), 3.98 (dt, .sup.3J.sub.2,3=3.3 Hz, .sup.3J.sub.2,OH=1.6 Hz, 1H; H-2), 3.72 (dd, .sup.3J.sub.3,4=9.6 Hz, 1H; H-3), 3.36 (s, 3H; OCH.sub.3), 2.39 (d, 1H; OH), 1.44 ppm (d, 3H; H-6); .sup.13C NMR (126 MHz, CDCl.sub.3): δ 137.2, 128-129, 100.0, 78.3, 72, 67.2, 66.4, 63.9, 55.0, 18.4 ppm; HRMS (ESI): m/z calcd for C.sub.14H.sub.19N.sub.3NaO.sub.4 [M+Na].sup.+: 316.12678. found: 316.12705.

1,2-Di-O-acetyl-4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (5)

(77) A solution of 4 (1.03 g, 5.07 mmol) in acetic anhydride/acetic acid/sulfuric acid (50:20:0.5, 35 mL) was stirred for 1.5 h at room temperature, and then poured into ice-cold 1 M K.sub.2CO.sub.3 solution. The product was extracted with dichloromethane and the extract was dried over MgSO.sub.4. The solvent was evaporated and co-evaporated with toluene. A chromatography column on silica (hexane/ethyl acetate 10:1) gave 5 as an anomeric mixture (1.4 g, 82%, α/β 93:7). The NMR parameters are in agreement with the literature (Boschiroli et al. (2001) Curr. Op. Microbiol. 4, 58-64): .sup.1H NMR (500 MHz, CDCl.sub.3): δ(α) 7.4-7.3 (m, 5H; H—Ar), 6.02 (d, .sup.3J.sub.1,2=2 Hz, 1H; H-1), 5.34 (dd, .sup.3J.sub.2,3=3.3 Hz, 1H; H-2), 2.15, 2.11 (2s, 6H; OAc), 1.34 ppm (d, 3H; H-6); .sup.13C NMR (126 MHz, CDCl.sub.3): δ 169.8, 168.3, 137.0, 128-129, 91.0, 75.8, 71.8, 69.3, 66.3, 63.6, 20.9, 20.8, 18.5 ppm; elemental analysis calcd (%) for C.sub.17H.sub.21N.sub.3O.sub.6: C, 56.2; H, 5.8; N, 11.7. found: C, 56.2; H, 5.7; N, 11.3.

1,2,3-Tri-O-acetyl-4-azido-4,6-dideoxy-α-D-mannopyranoside (6)

(78) Compound 3 (1.03 g, 5.07 mmol) was acetylated with the same protocol used to obtain compound 5. Compound 6 (1.31 g, 82%) was obtained in a a/l 95:5 mixture. The separation was done only for analysis: The NMR parameters and physical constants are in agreement with the literature (Bundle et al. (1988) Carbohydr. Res. 174, 239-251): [α].sub.D.sup.20(α)=+122 (c=1.2 in CHCl.sub.3); .sup.1H NMR (500 MHz, CDCl.sub.3): δ(α) 5.98 (d, .sup.3J.sub.1,2=1.6 Hz, 1H; H-1), 2.15, 2.13, 2.07 (3s, 9H; OAc), 1.36 ppm (d, 3H; H-6); .sup.13C NMR (126 MHz, CDCl.sub.3): δ 169.7, 169.6, 168.3, 90.6, 70.1, 69.3, 67.8, 62.1, 20.9, 20.7, 20.7, 18.4 ppm; HRMS (ESI): m/z calcd for C.sub.12H.sub.17N.sub.3NaO.sub.7 [M+Na].sup.+: 338.09587. found: 338.09559; elemental analysis calcd (%) for C.sub.12H.sub.17N.sub.3O.sub.7: C, 45.7; H, 5.4; N, 13.3. found: C, 45.35; H, 5.45; N, 13.2.

Allyl 2, 3-di-O-acetyl-4-azido-4,6-dideoxy-α-D-mannopyranoside (7)

(79) To a solution of 6 (900 mg, 2.85 mmol) in dichloromethane (4 mL), was added BF.sub.3.Et.sub.2O (0.4 mL, 3.24 mmol). The mixture was stirred for 1 h at room temperature before adding allylic alcohol (0.3 mL, 4.41 mmol) and then stirred again for 2 days. Once the reaction was done, the mixture was cooled to 0° C., a satd. NaHCO.sub.3 solution was added and the mixture was stirred for 30 min. The product was extracted with ethyl acetate, the extract was dried (MgSO.sub.4), filtrated and concentrated. Purification on column chromatography (hexane/ethyl acetate 10:1) gave the allyl glycoside 7 (625 mg, 70%): [α].sub.D.sup.20=+103 (c=1.5 in CHCl.sub.3); .sup.1H NMR (500 MHz, CDCl.sub.3): δ 5.25 (dd, .sup.3J.sub.2,3=3.6 Hz, .sup.3J.sub.3,4=10 Hz, 1H; H-3), 5.23 (dd, .sup.3J.sub.1,2=1.7 Hz, 1H; H-2), 4.76 (d, .sup.3J.sub.1,2=1.7 Hz, 1H; H-1), 2.15, 2.08 (2s, 6H; OAc), 1.37 ppm (d, 3H; H-6); .sup.13C NMR (126 MHz, CDCl.sub.3): δ 169.9, 169.6, 133.1, 118.1, 96.5, 70.4, 69.2, 68.4, 67.0, 62.7, 20.9, 20.8, 18.3 ppm; elemental analysis calcd (%) for C.sub.13H.sub.19N.sub.3O.sub.6: C, 49.8; H, 6.1; N, 13.4. found: C, 49.9; H, 6.3; N, 13.5.

Allyl 4-azido-4,6-dideoxy-α-D-mannopyranoside (8)

(80) Allyl glycoside 7 (625 mg, 2 mmol) in methanol (20 mL) was treated with a 0.1 M solution of sodium methoxide (0.3 mL). After 1 h, the reaction was complete and neutralized with ion exchange resin H.sup.+. Filtration and removal of the solvent under vacuum gave pure diol 8 (447 mg, 98%): [α].sub.D.sup.20=+117 (c=1.1 in CHCl.sub.3); .sup.1H NMR (600 MHz, CDCl.sub.3): δ 4.84 (d, .sup.3J.sub.1,2=1.7 Hz, 1H; H-1), 3.93 (dd, .sup.3J.sub.2,3=3.4 Hz, 1H; H-2), 1.34 ppm (d, 3H; H-6); .sup.13C NMR (126 MHz, CDCl.sub.3): δ 133.4, 117.7, 98.5, 70.5, 70.2, 68.2, 66.8, 66.0, 18.3 ppm (C-6); HRMS (ESI): m/z calcd for C.sub.9H.sub.15N.sub.3NaO.sub.4 [M+Na].sup.+: 252.09548. found: 252.09579.

Allyl 2-O-acetyl-4-azido-4,6-dideoxy-α-D-mannopyranoside (9)

(81) To a solution of diol 8 (77 mg, 0.34 mmol) in dichloromethane (3.5 mL) were added triethyl orthoacetate (0.6 mL, 3.3 mmol) and p-toluenesulfonic acid (5 mg, 0.03 mmol). The mixture was stirred for 3 hrs at 50° C. When complete, the reaction was neutralized by triethylamine and concentrated. Acetic acid (80%, 8 mL) was added and the mixture was stirred for 30 min at room temperature, then concentrated. Compound 9 (91 mg, 98%) was purified by chromatography on silica (ethyl acetate/hexanes 1:10): [α].sub.D.sup.20=+78 (c=1.0 in CHCl.sub.3); .sup.1H NMR (500 MHz, CDCl.sub.3): δ 5.08 (dd, .sup.3J.sub.1,2=1.7 Hz, .sup.3J.sub.2,3=3.6 Hz, 1H; H-2), 4.80 (d, 1H; H-1), 2.15 (s, 3H; OAc), 1.35 ppm (d, 3H; H-6); .sup.13C NMR (126 MHz, CDCl.sub.3): (170.8, 133.2, 117.9, 96.5, 71.7, 69.2, 68.3, 66.9, 66.0, 21.0, 18.3 ppm; elemental analysis calcd (%) for C.sub.11H.sub.17N.sub.3O.sub.5: C, 48.7; H, 6.3; N, 15.5. found: C, 48.8; H, 6.2; N, 15.3.

2-O-Acetyl-4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranose (10)

(82) To a solution of 5 (484 mg, 1.33 mmol) in dichloromethane (3 mL), dimethylamine (2 M in THF, 1.8 mL, 3.6 mmol) was added dropwise. The solution was then stirred for 2 days at room temperature. After evaporation of the solvent, pure compound 10 (432 mg) was obtained quantitatively: .sup.1H NMR (500 MHz, CDCl.sub.3): δ 7.2-7.4 (m, 5H; H—Ar), 5.30 (s, 1H; H-1 β), 5.17 (d, 1H; H-1 α), 2.19 (s, 3H; OAc β), 2.13 (s, 3H; OAc α), 1.38 (d, 3H; H-6 β), 1.33 ppm (d, 3H; H-6 α); .sup.13C NMR (126 MHz, CDCl.sub.3): δ 170.8, 170.3, 137.2, 136.8, 128-129, 92.9, 92.5, 78.5, 75.6, 71.7, 71.6, 71.2 (C-5 β), 68.5, 67.7, 67.0, 64.0, 63.4, 21.0, 20.9, 18.65, 18.5 ppm (C-6 β); elemental analysis calcd (%) for C.sub.15H.sub.19N.sub.3O.sub.5: C, 56.1; H, 6.0; N, 13.1. found: C, 56.0; H, 6.0; N, 13.0.

2-O-Acetyl-4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl N-phenyltrifluoroacetimidate (11)

(83) Compound 10 (1.04 g, 3.2 mmol) was dissolved in dry dichloromethane (30 mL). N-phenyl trifluoroacetimidoyl chloride (1.2 mL, 9.6 mmol) and Cs.sub.2CO.sub.3 (3.2 g, 9.6 mmol) were added and the mixture was stirred overnight at room temperature. After filtration through celite, compound 11 (1.4 g, 87%) was purify on silica gel column (hexane/ethyl acetate 10:1): .sup.1H NMR (500 MHz, CDCl.sub.3): δ 7.4-6.8 (m, 10H; H—Ar), 6.2 (br s, 1H; H-1 α), 5.85 (br s, 1H; H-2 α), 5.8 (br s, 1H; H-1 β), 5.46 (br s, 1H; H-2 β), 2.2 (s, 3H; OAc β), 2.15 (s, 3H; OAc α), 1.44 (d, .sup.3J.sub.5,6=6.0 Hz, 3H; H-6 α), 1.33 ppm (d, .sup.3J.sub.5,6=6.1 Hz, 3H; H-6 β); HRMS (ESI): m/z calcd for C.sub.23H.sub.23F.sub.3N.sub.4NaO.sub.5 [M+Na].sup.+: 515.15128. found: 515.15147.

Ethyl 2-O-acetyl-4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-1-thio-α-D-mannopyranoside (14)

(84) Donor 11 (3.44 g, 7 mmol) and acceptor 13 (obtained from 5 via 12) (Peters & Bundle (1989) Can. J. Chem. 67, 491-496) (1.75 g, 5.4 mmol) were dissolved in dry dichloromethane (50 mL) and TMSOTf (0.1 mL, 0.55 mol) was added at 0° C. The reaction was complete after 30 min. of stirring at 0° C., then 30 min. at room temperature and was quenched with few drops of NEt.sub.3. Disaccharide 14 (3.19 g, 94%) was obtained pure after flash chromatography column (toluene/ethyl acetate 1:0, then 9:1). The NMR parameters and physical constants are in agreement with the literature (Crump et al. (2003) Emerg. Infect. Dis. 9, 539-544): [α].sub.D.sup.20=+129 (c=1.2 in CHCl.sub.3); .sup.1H NMR (500 MHz, CDCl.sub.3): δ 7.4-7.2 (m, 10H; H—Ar), 5.39 (dd, .sup.3J.sub.1,2=1.6 Hz, .sup.3J.sub.2,3=3.2 Hz, 1H; H-2.sup.II), 5.16 (d, .sup.3J.sub.1,2=1.1 Hz, 1H; H-1.sup.I), 4.81 (d, 1H; H-1.sup.II), 3.89 (dd, .sup.3J.sub.2,3=2.9 Hz, 1H; H-2.sup.I), 2.53 (2qd, .sup.2J=13 Hz, .sup.3J=7.4 Hz, 2H; S—CH.sub.2—CH.sub.3), 2.10 (s, 3H; Ac), 1.30 (d, 3H; H-6.sup.II), 1.29 (d, 3H; H-6), 1.25 ppm (t, 3H; S—CH.sub.2—CH.sub.3); .sup.13C NMR (125 MHz, CDCl.sub.3): δ 170.0, 137.5, 137.2, 129-128, 99.7, 83.4, 78.2, 76.4, 75.5, 72.3, 71.7, 67.8, 67.7, 67.4, 64.5, 64.0, 25.7, 21.1, 18.6, 18.6, 15.0 ppm; elemental analysis calcd (%) for C.sub.30H.sub.38N.sub.6O.sub.7S: C, 57.5; H, 6.1; N, 13.4; S, 5.1. found: C, 57.5; H, 6.2; N, 13.2; S, 5.2.

Allyl 2-O-acetyl-4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→3) 2-O-acetyl-4-azido-4,6-dideoxy-α-D-mannopyranoside (15)

(85) Donor 14 (188 mg, 0.3 mmol) and acceptor 9 (54 mg, 0.2 mmol) were dissolved in dry dichloromethane (6 mL) with molecular sieves, then NIS (72 mg, 0.32 mmol) and trifluoromethanesulfonic acid (9 μL, 0.1 mmol) were added at −30° C. The reaction was stirred at this temperature for 5 hours and then filtered through celite. The mixture was washed with Na.sub.2S.sub.2O.sub.3 then KHCO.sub.3. Trisaccharide 15 (114 mg, 65%) was obtained pure after flash chromatography (hexane/ethyl acetate 10:1): [α].sub.D.sup.20=+64 (c=1.3 in CHCl.sub.3); 1H NMR (600 MHz, CDCl.sub.3): δ 7.2-7.4 (m, 10H; H—Ar), 5.05 (dd, .sup.3J.sub.1,2=1.8 Hz, .sup.3J.sub.2,3=3.5 Hz, 1H; H-2.sup.I), 4.97 (d, .sup.3J.sub.1,2=1.9 Hz, 1H; H-1.sup.II), 4.9 (d, 1H; H-1.sup.III), 4.76 (d, 1H; H-1.sup.I), 4.01 (dd, .sup.3J.sub.2,3=3.0 Hz, 1H; H-2.sup.II), 2.10, 2.09 (2s, 6H; OAc), 1.33 (d, .sup.3J.sub.5,6=6.0 Hz, 3H; H-6.sup.I), 1.32 (d, .sup.3J.sub.5,6=6.6 Hz, 3H; H-6.sup.III), 1.26 ppm (d, 3H; H-6.sup.II); .sup.13C NMR (126 MHz, CDCl.sub.3): δ 170.0, 169.8, 137.5, 137.1, 133.2, 127-129, 117.9, 101.0, 99.4, 96.2, 77.2, 77.1, 75.4, 73.3, 71.8, 71.6, 70.9, 68.5, 68.2, 67.8, 67.1, 66.9, 64.5, 63.8, 63.7, 21.0, 20.9, 18.5, 18.4 ppm; HRMS (ESI): m/z calcd for C.sub.39H.sub.49N.sub.9NaO.sub.12 [M+Na].sup.+: 858.33929. found: 858.33904.

2-O-Acetyl-4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→3) 2-O-acetyl-4-azido-4,6-dideoxy-α-D-mannopyranose (16)

(86) Allyl glycoside 15 (76 mg, 91 μmol) was dissolved in a solution of AcONa in AcOH/H.sub.2O 9:1 (0.2 M, 2 mL) and PdCl.sub.2 (32 mg, 180 μmol) was added. The mixture was stirred overnight at room temperature and neutralized with NaHCO.sub.3. The product was extracted with dichloromethane and washed with water. Chromatography on silica gel (hexane/ethyl acetate 6:1) gave hemiacetal 16 (54 mg, 73%): .sup.1H NMR (600 MHz, CDCl.sub.3): δ 7.2-7.4 (m, 10H; H—Ar), 5.4 (dd, .sup.3J.sub.1,2=1.9 Hz, .sup.3J.sub.2,3=3.2 Hz, 1H; H-2.sup.III), 5.14 (br s, 1H; H-1.sup.I), 5.08 (dd, 3J.sub.1,2=1.9 Hz, .sup.3J.sub.2,3=3.3 Hz, 1H; H-2.sup.I), 4.98 (d, .sup.3J.sub.1,2=2.0 Hz, 1H; H-1.sup.II), 4.9 (d, 1H; H-1.sup.III), 2.11, 2.09 (2s, 6H; OAc), 1.33 (d, 3H; H-6.sup.I), 1.32 (d, 3H; H-6.sup.III), 1.26 ppm (d, 3H; H-6.sup.II); .sup.13C NMR (126 MHz, CDCl.sub.3): δ 170.1, 169.8, 137.5, 137.1, 127-129, 101.0, 99.4, 91.8, 77.2, 76.6, 75.4, 73.4, 71.8, 71.6, 71.2, 68.2, 67.8, 67.2, 66.9, 64.5, 63.8, 63.7, 21.0, 20.9, 18.5, 18.5, 18.4 ppm; HRMS (ESI): m/z calcd for C.sub.36H.sub.45N.sub.9NaO.sub.2[M+Na].sup.+: 818.30799. found: 818.30635.

2-O-Acetyl-4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→3) 2-O-acetyl-4-azido-4,6-dideoxy-α-D-mannopyranosyl N-phenyltrifluoroacetimidate (17)

(87) Compound 17 (147 mg, 79%) was prepared from trisaccharide 16 (153 mg, 192 μmol) as described for 11 and obtained as a mixture α/β 3:2: .sup.1H NMR (600 MHz, CDCl.sub.3): δ(α) 7.4-6.8 (m, 15H; H—Ar), 6.12 (br s, 1H; H-1.sup.I), 5.63 (br s, 1H; H-2.sup.I), 5.39 (dd, .sup.3J.sub.1,2=1.9 Hz, .sup.3J.sub.2,3=3.1 Hz, 1H; H-2.sup.I), 4.97 (br s, 1H; H-1.sup.III), 4.89 (d, 1H; H-1.sup.III), 4.0 (br s, 1H; H-2.sup.II), 2.16, 2.08 (2s, 6H; OAc), 1.46 (d, .sup.3J.sub.5,6=6.2 Hz, 3H; H-6.sup.I), 1.29 (d, .sup.3J.sub.5,6=6.2 Hz, 3H; H-6.sup.III), 1.26 ppm (d, .sup.3J.sub.5,6=6.2 Hz, 3H; H-6.sup.II); HRMS (ESI): m/z calcd for C.sub.44H.sub.49F.sub.3N.sub.10NaO.sub.12 [M+Na].sup.+: 989.33757. found: 989.33761.

2-O-Acetyl-4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranose (18)

(88) To a solution of disaccharide thioglycoside 14 (210 mg, 0.33 mmol) in acetone (6 mL), NIS (90 mg, 0.44 mmol) and water (100 μL) were added at 0° C. and stirred at this temperature for 30 min. and then overnight at room temperature. After completion, NaHCO.sub.3 solid was added and acetone evaporated. An extraction dichloromethane/water and then a purification on silica gel gave the hemiacetal 18 (144 mg, 76%): H NMR (600 MHz, CDCl.sub.3): δ(α) 7.4-7.2 (m, 10H; H—Ar), 5.42 (dd, .sup.3J.sub.1,2=2 Hz, .sup.3J.sub.2,3=3.2 Hz, 1H; H-2.sup.II), 5.12 (dd, .sup.3J.sub.1,2=1.8 Hz, .sup.3J.sub.1,OH=3.5 Hz, 1H; H-1.sup.I), 4.87 (d, 1H; H-1.sup.II), 3.89 (dd, .sup.3J.sub.2,3=3 Hz, 1H; H-2.sup.I), 2.10 (s, 3H; Ac), 1.30 ppm (2d, 6H; H-6.sup.I, H-6.sup.II); .sup.13C NMR (126 MHz, CDCl.sub.3): δ(α) 169.9, 137.7, 137.2, 129-127, 99.5, 93.6, 77.3, 75.5, 74.1, 72.3, 71.7, 67.7, 67.4, 67.3, 64.3, 64.0, 21.1, 18.8, 18.7 ppm; elemental analysis calcd (%) for C.sub.28H.sub.34N.sub.6O.sub.8: C, 57.7; H, 5.9; N, 14.4. found: C, 57.9; H, 6.0; N, 14.0.

2-O-Acetyl-4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl N-phenyltrifluoroacetimidate (19)

(89) Disaccharide 18 (538 mg, 0.92 mmol) was dissolved in dry dichloromethane (9 mL), N-phenyl trifluoroacetimidoyl chloride (350 μL, 2.8 mmol) and Cs.sub.2CO.sub.3 (0.9 g, 2.8 mmol) were added and the mixture was stirred overnight at room temperature. After evaporation of the solvent chromatography on silica gel (toluene/ethyl acetate 1:0 to 10:2) gave pure imidate 19 (623 mg, 90%): .sup.1H NMR (500 MHz, CDCl.sub.3): δ(α) 7.4-6.7 (m, 15H; H—Ar), 6.05 (br s, 1H; H-1.sup.I), 5.41 (br s, 1H; H-2.sup.II), 4.86 (br s, 1H; H-1.sup.II), 3.85 (br s, 1H; H-2.sup.I), 2.10 (s, 3H; Ac), 1.33 (d, .sup.3J.sub.5,6=6.2 Hz, 3H; H-6.sup.I), 1.18 ppm (d, .sup.3J.sub.5,6=6.2 Hz, 3H; H-6.sup.II); .sup.19F NMR (469 MHz, CDCl.sub.3): δ(α) −75.7 ppm; .sup.13C NMR (125 MHz, CDCl.sub.3): δ 170.0, 143.3, 137.3, 137.2, 129-128, 124.7, 119.5, 99.5, 95.7, 77.1, 75.5, 72.9, 72.2, 71.8, 70.3, 68.0, 67.2, 63.9, 63.5, 21.1, 18.7, 18.5 ppm; HRMS (ESI): m/z calcd for C.sub.36H.sub.38F.sub.3KN.sub.7O.sub.8[M+K].sup.+: 792.2366. found: 792.2363.

5-Methoxycarbonylpentyl 2-O-acetyl-4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (21)

(90) Donor 19 (110 mg, 0.15 mmol) and 5-methoxycarbonylpentanol 20 (El Fangour et al. (2004) J. Org. Chem. 69, 2498-2503) (30 mg, 0.2 mmol) in solution in toluene (7 mL) with some molecular sieves were heated at 100° C. and TMSOTf (2 μL, 10 μmol) was added. After heating 1 hour at this same temperature, the reaction was quenched with pyridine, and the mixture filtered. Compound 21 (59 mg, 53%) was obtained after purification on silica gel chromatography column (eluent: ethyl acetate/hexane 1/6): [α].sub.D.sup.20=+78 (c=1.0 in CHCl.sub.3); .sup.1H NMR (500 MHz, CDCl.sub.3): δ 7.2-7.4 (m, 10H; H—Ar), 5.41 (dd, 1H, .sup.3J.sub.1,2=1.9 Hz, .sup.3J.sub.2,3=3.2 Hz, H-2.sup.II), 4.86 (d, 1H, .sup.3J.sub.1,2=1.8 Hz, H-1), 4.66 (d, .sup.3J.sub.1,2=1.8 Hz, 1H; H-1.sup.I), 3.84 (dd, .sup.3J.sub.1,2=2 Hz, .sup.3J.sub.2,3=2.8 Hz, 1H; H-2.sup.I), 2.33 (t, .sup.3J.sub.d,e=7.5 Hz, 2H; H-e), 2.09 (s, 3H; Ac), 1.65 (quint, .sup.3J.sub.c,d=.sup.3J.sub.d,e=7.6 Hz, 2H; H-d), 1.57 (m, 2H; H-b), 1.36 (m, 2H; H-c), 1.31 (d, .sup.3J.sub.5,6=6 Hz, 3H; H-6.sup.II), 1.29 ppm (d, .sup.3J.sub.5,6=6 Hz, 3H; H-6.sup.I); 1.sup.3C NMR (125 MHz, CDCl.sub.3): δ 174.1, 169.9, 137.7, 137.2, 129-128, 99.5, 98.7, 77.9, 75.5, 74.1, 72.2, 71.7, 67.7, 67.7, 67.4, 67.2, 64.3, 64.0, 51.6, 34.1, 29.2, 25.8, 24.8, 21.1, 18.7, 18.6 ppm; elemental analysis calcd (%) for C.sub.35H.sub.46N.sub.6O.sub.10: C, 59.1; H, 6.5; N, 11.8. found: C, 59.0; H, 6.6; N, 11.4.

5-Methoxycarbonylpentyl 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (22)

(91) Compound 21 (70 mg, 98 μmol) in methanol (3 mL) was treated with a 0.1 M solution of sodium methoxide (0.1 mL). After 1 h, the reaction was complete and neutralized with ion exchange resin H.sup.+. Filtration and removal of the solvent under vacuum gave quantitatively pure acceptor 22 (65 mg): [α].sub.D.sup.20=+90 (c=1.7 in CHCl.sub.3); .sup.1H NMR (500 MHz, CDCl.sub.3): δ 7.3-7.4 (m, 10H; H—Ar), 4.94 (d, .sup.3J.sub.1,2=1.7 Hz, 1H; H-1.sup.II), 4.67 (d, .sup.3J.sub.1,2=2.1 Hz, 1H; H-1.sup.I), 3.99 (dd, .sup.3J.sub.2,3=3.1 Hz, 1H; H-2.sup.II), 3.84 (dd, .sup.3J.sub.2,3=3.0 Hz, 1H; H-2.sup.I), 3.35 (dt, .sup.2J=9.7 Hz, .sup.3J.sub.a,b=6.4 Hz, 1H; H-a), 2.33 (t, .sup.3J.sub.d,e=7.5 Hz, 2H; H-e), 1.65 (quint, .sup.3J.sub.c,d=7.6 Hz, 2H; H-d), 1.57 (m, 2H; H-b), 1.36 (m, 2H; H-c), 1.30 (d, .sup.3J.sub.5,6=6.2 Hz, 3H; H-6.sup.II), 1.30 ppm (d, 3H; H-6.sup.I); .sup.13C NMR (125 MHz, CDCl.sub.3): δ 174.1, 137.6, 137.3, 129-128, 101.0, 98.9, 78.0, 77.8, 74.1, 72.3, 72.2, 67.7, 67.4, 67.3, 67.2, 64.5, 64.0, 51.6, 34.1, 29.2, 25.8, 24.8, 18.7, 18.6 ppm; elemental analysis calcd (%) for C.sub.33H.sub.44N.sub.6O.sub.9: C, 59.3; H, 6.6; N, 12.6. found: C, 59.2; H, 6.45; N, 12.2.

5-Methoxycarbonylpentyl 2-O-acetyl-4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→3) 2-O-acetyl-4-azido-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (23)

(92) Trisaccharide imidate 17 (45 mg, 47 mol) and disaccharide alcohol 22 (17 mg, 25 μmol) were dissolved in dichloromethane (1 mL) and TMSOTf (1 μL, 5 μmol) was added at 0° C. The mixture was stirred 30 min. at 0° C. and 1 hour at room temperature before being quenched with one drop of pyridine. Purification on silica gel column (eluent: ethyl acetate/hexane 1/10) gave pure pentasaccharide 23 (25 mg, 68%): [α].sub.D.sup.20=+80 (c=1.0 in CHCl.sub.3); 1H NMR (500 MHz, CDCl.sub.3): δ 7.2-7.4 (m, 20H; H—Ar), 5.41 (dd, .sup.3J.sub.1,2=1.7 Hz, .sup.3J.sub.2,3=3.2 Hz, 1H; H-2.sup.V), 5.13 (dd, .sup.3J.sub.1,2=1.9 Hz, .sup.3J.sub.2,3=3.2 Hz, 1H; H-2.sup.III), 5.00 (d, .sup.3J.sub.1,2=1.7 Hz, 1H; H-1.sup.II), 4.95 (d, .sup.3J.sub.1,2=1.5 Hz, 1H; H-1.sup.IV), 4.90 (d, 1H; H-1.sup.V), 4.82 (d, 1H; H-1.sup.III), 4.61 (br s, 1H; H-1.sup.I), 3.98 (dd, .sup.3J.sub.2,3=2.9 Hz, 1H; H-2.sup.II), 3.84 (dd, .sup.3J.sub.2,3=2.3 Hz, 1H; H-2.sup.IV), 3.81 (dd, .sup.3J.sub.2,3=2.3 Hz, 1H; H-2.sup.I), 3.68 (s, 3H; H-g), 3.58 (dt, .sup.2J=9.7 Hz, .sup.3J.sub.a,b=6.6 Hz, 1H; H-a), 3.34 (dt, .sup.3J.sub.a,b=6.4 Hz, 1H; H-a), 2.32 (t, .sup.3J.sub.d,e=7.5 Hz, 2H; H-e), 2.1 (s, 6H; Ac), 1.65 (quint, .sup.3J.sub.c,d=7.6 Hz, 2H; H-d), 1.55 (m, 2H; H-b), 1.34 (m, 2H; H-c), 1.30 (d, 3H; H-6.sup.V), 1.29 (d, 3H; H-6.sup.IV), 1.28 (d, 3H; H-6.sup.I), 1.25 (d, 3H; H-6.sup.II), 1.18 ppm (d, 3H; H-6.sup.III); .sup.13C NMR (125 MHz, CDCl.sub.3): δ 174.1, 169.9, 169.6, 137.7, 137.5, 137.5, 137.2, 129-128, 100.8, 100.3, 99.6, 98.8, 98.8, 77.8, 77.4, 76.8, 76.1, 75.5, 74.3, 73.9, 73.7, 72.3, 72.2, 72.0, 71.8, 70.6, 67.6, 68.4, 67.9, 67.9, 67.8, 67.3, 67.2, 64.6, 64.6, 64.2, 64.0, 63.9, 51.7, 34.1, 29.2, 25.8, 24.8, 21.1, 21.0, 18.8, 18.7, 18.6, 18.5, 18.4 ppm; HRMS (ESI): m/z calcd for C.sub.69H.sub.7N.sub.15NaO.sub.20 [M+Na].sup.+: 1468.6144. found: 1468.6140.

5-Methoxycarbonylpentyl 4-azido-3-O-benzyl-4,6-Dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→3) 4-azido-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (24)

(93) Pentasaccharide 23 (82 mg, 57 μmol) in methanol (3 mL) was treated with a 0.1 M solution of sodium methoxide (0.1 mL). The reaction was stirred 1 hour at room temperature and then neutralized with ion exchange resin H.sup.+. Filtration, evaporation and column chromatography (eluent: ethyl acetate/toluene 1:6) gave pure compound 24 (67 mg, 86%): [α].sub.D.sup.20=+80 (c=1.1 in CHCl.sub.3); .sup.1H NMR (500 MHz, CDCl.sub.3): δ 7.2-7.4 (m, 20H; H—Ar), 5.04 (d, .sup.3J.sub.1,2=1.8 Hz, 1H; H-1.sup.IV), 4.98 (d, .sup.3J.sub.1,2=1.7 Hz, 1H; H-1.sup.II), 4.95 (d, .sup.3J.sub.1,2=1.8 Hz, 1H; H-1.sup.V), 4.86 (d, .sup.3J.sub.1,2=1.7 Hz, 1H; H-1.sup.III), 4.62 (br s, 1H; H-1.sup.I), 4.06 (dd, .sup.3J.sub.2,3=2.9 Hz, 1H; H-2.sup.IV), 3.99 (dd, .sup.3J.sub.2,3=3.1 Hz, 1H; H-2.sup.II), 3.94 (dd, .sup.3J.sub.2,3=2.9 Hz, 1H; H-2.sup.V), 3.93 (dd, .sup.3J.sub.2,3=3.1 Hz, 1H; H-2.sup.III), 3.83 (app. t, .sup.3J.sub.1,2=3J.sub.2,3=2.9 Hz, 1H; H-2.sup.I), 3.68 (s, 3H; H-g), 3.34 (dt, .sup.3J.sub.a,b=6.4 Hz, 1H; H-a), 3.24 (t, 1H; H-4.sup.I), 2.32 (t, .sup.3J.sub.d,e=7.5 Hz, 2H; H-e), 1.65 (quint, .sup.3J.sub.c,d=7.6 Hz, 2H; H-d), 1.56 (m, 2H; H-b), 1.34 (m, 2H; H-c), 1.32 (d, 3H; H-6.sup.II), 1.29 (d, 6H; H-6.sup.IV, H-6V), 1.29 (d, 3H; H-6.sup.I), 1.17 ppm (d, 3H; H-6.sup.III); .sup.13C NMR (125 MHz, CDCl.sub.3): δ 174.1, 137.5, 137.5, 137.4, 137.3, 129-128, 101.1, 100.8, 100.7, 100.5, 98.8, 79.0, 77.9, 77.8, 77.8, 77.3, 73.8, 73.5, 73.4, 72.4, 73.4, 72.3, 72.3, 69.6, 67.7, 68.4, 67.9, 67.6, 67.6, 67.3, 67.3, 64.6, 64.4, 64.0, 63.9, 63.9, 51.7, 34.1, 29.2, 25.8, 24.8, 18.7, 18.7, 18.7, 18.5, 18.4 ppm; HRMS (ESI): m/z calcd for C.sub.65H.sub.83N.sub.15NaO.sub.18 [M+Na].sup.+: 1384.5933. found: 1384.5926.

5-Methoxycarbonylpentyl 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→3) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranoside (1)

(94) A solution of pentasaccharide 24 (67 mg, 49 μmol) in a mixture pyridine/NEt.sub.3 1:1 (6 mL) was saturated with H.sub.2S for 1 h and the media was then stirred for 24 h at room temperature. The solvent was co-evaporated with toluene and mass spectrometry of the crude product showed only one peak corresponding to compound 25 and no products arising from incomplete reduction: HRMS (ESI): m/z calcd for C.sub.65H.sub.94N.sub.5O.sub.18 [M+H].sup.+: 1232.6588. found: 1232.6577. This crude material was directly used for formylation.

(95) Compound 25 was dissolved in methanol (5 mL) and a solution of acetic anhydride/formic acid 2:1 (0.5 mL) was added. The mixture was stirred at room temperature overnight. The solvent was evaporated and pentasaccharide 26 (43 mg, 63% over 2 steps) was purified with a chromatography column (eluent: MeOH/DCM 1:10): HRMS (ESI): m/z calcd for C.sub.70H.sub.93N.sub.5NaO.sub.23 [M+Na].sup.+: 1394.6154. found: 1394.6151.

(96) Compound 26 (43 mg, 31 μmol) in solution in acetic acid (8 mL) with palladium on charcoal was stirred overnight at room temperature under atmosphere of hydrogen. After filtration and concentration, pentasaccharide 1 (17 mg, 54%) was purified on a reverse phase HPLC column (MeCN/H.sub.2O 15:85): .sup.1H NMR (600 MHz, D.sub.2O): δ 8.21-8.19 (Z) and 8.02-8.00 (E) (m, 5H; NCHO), 5.20-4.90 (m, 5H; 5×H-1), 3.72-3.66 (m, 4H, H-a, H-g), 3.56-3.50 (m, 1H; H-a), 2.40 (t, .sup.3J.sub.d,e=7.4 Hz, 2H; H-e), 1.65-1.56 (m, 4H; H-b, H-d), 1.41-1.32 (m, 2H; H-c), 1.30-1.18 ppm (m, 15H; 5×H-6); HRMS (ESI): m/z calcd for C.sub.42H.sub.69N.sub.5NaO.sub.23 [M+Na].sup.+: 1034.4276. found: 1034.4275.

Ethyl 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-1-thio-α-D-mannopyranoside (27)

(97) Disaccharide 14 (570 mg, 0.91 mmol) in methanol (10 mL) was treated with a 0.1 M solution of sodium methoxide (0.3 mL). The reaction was stirred 2 hours at room temperature and then neutralized with ion exchange resin H.sup.+. Filtration, evaporation and column chromatography (eluent: ethyl acetate/hexane 1:4) gave pure alcohol 27 (433 mg, 81%): [α].sub.D.sup.20=+175 (c=1.0 in CHCl.sub.3); .sup.1H NMR (600 MHz, CDCl.sub.3): δ 7.5-7.3 (m, 10H; H—Ar), 5.18 (d, .sup.3J.sub.1,2=1.4 Hz, 1H; H-1.sup.I), 4.91 (d, .sup.3J.sub.1,2=1.5 Hz, 1H; H-1.sup.II), 3.99 (ddd, .sup.3J.sub.2,3=3.2 Hz, .sup.3J.sub.2,OH=1.6 Hz, 1H; H-2.sup.II), 3.96 (dd, .sup.3J.sub.2,3=3 Hz, 1H; H-2.sup.I), 2.61 (dq, .sup.3J=7.4 Hz, .sup.2J=12.9 Hz, 1H; S—CH.sub.2), 2.55 (dq, .sup.3J=7.5 Hz, 1H; S—CH.sub.2), 1.30 (d, 6H; H-6.sup.I, H-6.sup.II), 1.27 ppm (t, 3H; S—CH.sub.2—CH.sub.3); .sup.13C NMR (126 MHz, CDCl.sub.3): δ 137.4, 137.2, 129-128, 101.1, 83.6, 78.3, 77.8, 76.1, 72.3, 72.3, 67.7, 67.5, 67.3, 64.7, 64.0, 25.8, 18.6, 18.6, 15.0 ppm; HRMS (ESI): m/z calcd for C.sub.28H.sub.36N.sub.6NaO.sub.6S [M+Na].sup.+: 607.2309. found: 607.2303.

Ethyl 2-O-acetyl-4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-1-thio-α-D-mannopyranoside (28)

(98) Donor 19 (226 mg, 0.30 mmol) and acceptor 27 (100 mg, 0.17 mmol) were dissolved in toluene (3 mL) with molecular sieves and TMSOTf (2 μL, 11 μmol) was added at 100° C. The mixture was stirred 1 hour at 100° C. before being quenched with one drop of pyridine. After filtration, a purification on silica gel column using toluene to elute the leaving group of the donor and then a mixture ethyl acetate/toluene 5:95 gave pure tetrasaccharide 28 (159 mg, 81%): [α].sub.D.sup.20=+122 (c=1.0 in CHCl.sub.3); .sup.1H NMR (600 MHz, CDCl.sub.3): δ 7.4-7.3 (m, 20H; H—Ar), 5.41 (dd, .sup.3J.sub.1,2=1.8 Hz, .sup.3J.sub.2,3=3.4 Hz, 1H; H-2.sup.IV), 5.10 (d, .sup.3J.sub.1,2=1.4 Hz, 1H; H-1.sup.II), 4.95 (d, .sup.3J.sub.1,2=1.8 Hz, 1H; H-1.sup.III), 4.85 (d, 1H; H-1.sup.V), 4.84 (d, .sup.3J.sub.1,2=1.8 Hz, 1H; H-1.sup.II), 3.88 (dd, .sup.3J.sub.2,3=2.9 Hz, 1H; H-2.sup.III), 3.85 (dd, .sup.3J.sub.2,3=2.9 Hz, 1H; H-2.sup.I), 3.83 (dd, .sup.3J.sub.2,3=2.9 Hz, 1H; H-2.sup.II), 2.59 (dq, .sup.3J=7.4 Hz, .sup.2J=13 Hz, 1H; S—CH.sub.2), 2.53 (dq, .sup.3J=7.5 Hz, 1H; S—CH.sub.2), 2.11 (s, 3H; Ac), 1.27 (2d, 6H; H-6.sup.I, H-6.sup.II), 1.25 (t, 3H; S—CH.sub.2—CH.sub.3), 1.21 (d, 3H; H-6.sup.IV), 1.20 ppm (d, 3H; H-6.sup.III); .sup.13C NMR (126 MHz, CDCl.sub.3): δ 169.9, 137.6, 137.3, 137.3, 137.2, 129-128, 100.7, 100.2, 99.3, 83.5 77.9, 77.0, 76.8, 76.2, 75.6, 73.6, 73.6, 72.4, 72.3, 72.2, 71.7 (CH.sub.2-Ph), 68.1, 68.0, 67.8, 67.7, 67.3, 64.7, 64.4, 64.2, 64.0, 25.7, 21.1, 18.8, 18.7, 18.6, 18.5, 15.0 ppm; HRMS (ESI): m/z calcd for C.sub.56H.sub.68N.sub.12NaO.sub.13S [M+Na].sup.+: 1171.4642. found: 1171.4644.

Allyl 2-O-acetyl-4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→3) 2-O-acetyl-4-azido-4,6-dideoxy-α-D-mannopyranoside (29)

(99) Donor 28 (197 mg, 171 mol) and acceptor 9 (37 mg, 136 mol) were dissolved in dry dichloromethane (5 mL) with molecular sieves, then NIS (46 mg, 200 mol) and trifluoromethanesulfonic acid (6 μL, 68 μmol) were added at 0° C. The reaction was stirred at this temperature for 15 min. and then filtered through celite. The mixture was washed with Na.sub.2S.sub.2O.sub.3 then KHCO.sub.3. Pentaccharide 29 (125 mg, 68%) was obtained pure after flash chromatography (hexane/ethyl acetate 6:1): [α].sub.D.sup.20=+88 (c=1.0 in CHCl.sub.3); .sup.1H NMR (600 MHz, CDCl.sub.3): δ 7.5-7.2 (m, 20H; H—Ar), 5.41 (dd, .sup.3J.sub.1,2=1.8 Hz, .sup.3J.sub.2,3=3.2 Hz, 1H; H-2.sup.V), 5.41 (dd, .sup.3J.sub.1,2=1.8 Hz, .sup.3J.sub.2,3=3.3 Hz, 1H; H-2.sup.I), 4.93 (m, 3H; H.sup.II, H-1.sup.III, H-1.sup.IV), 4.85 (d, 1H; H-1), 4.75 (d, 1H; H-1.sup.I), 3.87 (dd, .sup.3J.sub.1,2=1.8 Hz, .sup.3J.sub.2,3=2.9 Hz, 1H; H-2.sup.IV), 3.82 (dd, .sup.3J.sub.1,2=1.8 Hz, .sup.3J.sub.2,3=2.9 Hz, 1H; H-2.sup.III), .sup.32.10, 2.08 (2s, 6H; Ac), 1.33 (d, 3H; H-6.sup.I), 1.29 (d, .sup.3J.sub.5,6=6.2 Hz, 3H; H-6.sup.III), 1.24 (d, .sup.3J.sub.5,6=6.2 Hz, 3H; H-6.sup.II), 1.20 (d, .sup.3J.sub.5,6=6.2 Hz, 3H; H-6.sup.V), 1.14 ppm (d, .sup.3J.sub.5,6=6.2 Hz, 3H; H-6.sup.IV); .sup.13C NMR (126 MHz, CDCl.sub.3): δ 170.1, 169.9, 137.6, 137.4, 137.3, 137.3, 129-128, 133.3 118.1, 101.2, 100.5, 100.3, 99.2, 96.4, 77.4, 77.0, 76.8, 75.6, 73.6, 73.5, 73.3, 72.4, 72.2, 72.0, 71.7, 71.0, 68.6, 68.3, 68.2, 68.0, 67.8, 67.3, 67.1, 64.6, 64.4, 64.2, 64.1, 64.0, 21.1, 21.0, 18.7, 18.6, 18.6, 18.6, 18.5 ppm; HRMS (ESI): m/z calcd for C.sub.65H.sub.79N.sub.15NaO.sub.18 [M+Na].sup.+: 1380.562. found: 1380.5611.

2-O-Acetyl-4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→3) 2-O-acetyl-4-azido-4,6-dideoxy-α-D-mannopyranose (30)

(100) Allyl glycoside 29 (40 mg, 29 μmol) was dissolved in a solution of AcOH/H.sub.2O 9:1 (0.6 mL), AcONa (10 mg, 122 μmol) and PdCl.sub.2 (10 mg, 56 μmol) were added. The mixture was stirred overnight at room temperature and neutralized with NaHCO.sub.3. The product was extracted with dichloromethane, washed with water, dried over MgSO.sub.4, filtrated and concentrated. Chromatography on silica gel (hexane/ethyl acetate 2:1) gave compound 30 (24 mg, 62%): .sup.1H NMR (600 MHz, CDCl.sub.3): δ 7.4-7.25 (m, 20H; H—Ar), 5.40 (dd, .sup.3J.sub.1,2=1.8 Hz, .sup.3J.sub.2,3=3.2 Hz, 1H; H-2.sup.V), 5.13 (br s, 1H; H-1.sup.I), 5.06 (dd, .sup.3J.sub.1,2=1.8 Hz, .sup.3J.sub.2,3=3.3 Hz, 1H; H-2.sup.I), 4.94 (d, .sup.3J.sub.1,2=1.8 Hz, 1H; H-1.sup.II), 4.92 (d, .sup.3J.sub.1,2=1.7 Hz, 2H; H-1.sup.III, H-1.sup.IV), 4.84 (d, 1H; H-1.sup.V), 3.98 (dd, .sup.3J.sub.2,3=2.8 Hz, 1H; H-2.sup.II), 3.87 (dd, .sup.3J.sub.2,3=2.9 Hz, 1H; H-2.sup.IV), 3.82 (dd, .sup.3J.sub.2,3=2.8 Hz, 1H; H-2.sup.III), 2.10, 2.08 (2s, 6H; Ac), 1.33 (d, 3H; H-6.sup.I), 1.29 (d, .sup.3J.sub.5,6=6.2 Hz, 3H; H-6.sup.III), 1.24 (d, .sup.3J.sub.5,6=6.2 Hz, 3H; H-6.sup.II), 1.20 (d, .sup.3J.sub.5,6=6.2 Hz, 3H; H-6.sup.V), 1.14 ppm (d, .sup.3J.sub.5,6=6.2 Hz, 3H; H-6.sup.IV). .sup.13C NMR (126 MHz, CDCl.sub.3): δ 170.2, 170.0, 137.6, 137.4, 137.3, 137.3, 129-128, 101.2, 100.5, 100.3, 99.2, 92.0, 77.0, 76.8, 76.6, 76.6, 75.6, 73.6, 73.6, 73.4, 72.4, 72.2, 72.0, 71.7, 71.2, 68.3, 68.2, 68.0, 67.8, 67.3, 67.1, 64.6, 64.4, 64.2, 64.1, 64.0, 21.1, 21.0, 18.7, 18.6, 18.6, 18.6, 18.5 ppm. HRMS (ESI): m/z calcd for C.sub.62H.sub.75N.sub.15NaO.sub.18 [M+Na].sup.+: 1340.5307. found: 1340.5291.

2-O-Acetyl-4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→3) 2-O-acetyl-4-azido-4,6-dideoxy-α-D-mannopyranosyl N-phenyltrifluoroacetimidate (31)

(101) To a solution of free pentasaccharide 30 (118 mg, 89 μmol) in dichloromethane (2 mL) were added N-phenyl trifluoroacetimidoyl chloride (34 μL, 270 μmol) and Cs.sub.2CO.sub.3 (90 mg, 280 mol). The mixture was stirred overnight at room temperature. After filtration, a purification on silica gel column (eluent: ethyl acetate/hexane 1:9) gave donor 31 (104 mg, 79%) in a a/3 mixture which was used directly for the next glycosylation: HRMS (ESI): m/z calcd for C.sub.70H.sub.79F.sub.3N.sub.16NaO.sub.18 [M+Na].sup.+: 1511.5603. found: 1511.5600.

5-Methoxycarbonylpentyl 2-O-acetyl-4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (32)

(102) Donor 19 (117 mg, 160 μmol) and acceptor 22 (65 mg, 97 μmol) were dissolved in toluene (2 mL) with molecular sieves and TMSOTf (2 μL, 11 μmol) was added at 100° C. The mixture was stirred 1 hour at 100° C. before being quenched with one drop of pyridine. After filtration, a purification on silica gel column using toluene to elute the leaving group of the donor and then a mixture ethyl acetate/toluene 5:95 gave pure tetrasaccharide 32 (93 mg, 77%): [α]D.sup.20=+55 (c=1.0 in CHCl.sub.3); .sup.1H NMR (600 MHz, CDCl.sub.3): δ 7.4-7.3 (m, 20H; H—Ar), 5.40 (dd, .sup.3J.sub.1,2=1.8 Hz, .sup.3J.sub.2,3=3.2 Hz, 1H; H-2.sup.IV), 4.94 (d, .sup.3J.sub.1,2=1.8 Hz, 1H; H-1.sup.III), 4.87 (d, .sup.3J.sub.1,2=1.9 Hz, 1H; H-1.sup.II), 4.84 (d, 1H; H-1.sup.IV), 4.60 (d, .sup.3J.sub.1,2=1.5 Hz, 1H; H-1.sup.I), 3.87 (dd, .sup.3J.sub.2,3=3 Hz, 1H; H-2.sup.II), 3.83 (dd, .sup.3J.sub.2,3=2.9 Hz, 1H; H-2.sup.II), 3.78 (dd, .sup.3J.sub.2,3=2.9 Hz, 1H; H-2.sup.I), 3.68 (s, 3H; H-g), 3.57 (dt, .sup.3J.sub.a,b=6.6 Hz, .sup.2J=9.6 Hz, 1H; H-a), 3.32 (dt, .sup.3J.sub.a,b=6.4 Hz, 1H; H-a), 2.32 (t, .sup.3J.sub.d,e=7.5 Hz, 2H; H-e), 2.11 (s, 3H; Ac), 1.64 (quint, .sup.3J.sub.c,d=7.5 Hz, 2H; H-d), 1.55 (m, 2H; H-b), 1.34 (m, 2H; H-c), 1.26 (2d, 6H; H-6.sup.I, H-6.sup.II), 1.20 (d, 3H; H-6.sup.IV), 1.16 ppm (d, 3H; H-6.sup.III); .sup.13C NMR (151 MHz, CDCl.sub.3): δ 174.1, 169.9, 137.6, 137.5, 137.3, 137.3, 129-128, 100.6, 100.2, 99.2, 98.7, 77.6, 77.0, 76.8, 75.6, 74.2, 73.6, 73.6, 72.4, 72.3, 72.2, 71.7, 68.0, 67.9, 67.8, 67.7, 67.3, 67.2, 64.5, 64.4, 64.2, 64.0, 51.7, 34.1, 29.2, 25.8, 24.8, 21.1, 18.8, 18.7, 18.6, 18.5 ppm; elemental analysis calcd (%) for C.sub.61H.sub.76N.sub.12O.sub.16: C, 59.40; H, 6.21; N, 13.63. found: C, 59.62; H, 5.93; N, 13.36.

5-Methoxycarbonylpentyl 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (33)

(103) Tetrasaccharide 32 (90 mg, 73 mol) in methanol (3 mL) was treated with a 0.1 M solution of sodium methoxide (0.1 mL). The reaction was stirred 2 hours at room temperature and then neutralized with ion exchange resin H.sup.+. Filtration, evaporation and chromatography on silica gel (eluent: ethyl acetate/hexane 1:6) gave pure alcohol 33 (73 mg, 84%): [α].sub.D.sup.20=+104 (c=1.0 in CHCl.sub.3); .sup.1H NMR (600 MHz, CDCl.sub.3): δ 7.4-7.3 (m, 20H; H—Ar), 4.97 (d, .sup.3J.sub.1,2=1.8 Hz, 1H; H-1), 4.95 (d, .sup.3J.sub.1,2=1.9 Hz, 1H; H-1.sup.III), 4.87 (d, .sup.3J.sub.1,2=1.9 Hz, 1H; H-1.sup.II), 4.60 (d, .sup.3J.sub.1,2=1.8 Hz, 1H; H-1.sup.I), 5.40 (dd, .sup.3J.sub.2,3=3.0 Hz, 1H; H-2.sup.IV), 3.87 (dd, .sup.3J.sub.2,3=3.0 Hz, 1H; H-2.sup.III), 3.83 (dd, .sup.3J.sub.2,3=2.9 Hz, 1H; H-2.sup.II), 3.78 (dd, .sup.3J.sub.2,3=2.9 Hz, 1H; H-2.sup.I), 3.32 (dt, .sup.3J.sub.a,b=6.4 Hz, 1H; H-a), 2.32 (t, .sup.3J.sub.d,e=7.6 Hz, 2H; H-e), 2.30 (br. s, 1H; OH), 1.64 (quint, .sup.3J.sub.c,d=7.6 Hz, 2H; H-d), 1.55 (m, 2H; H-b), 1.34 (m, 2H; H-c), 1.26 (2d, 6H; H-6, H-6.sup.II), 1.20 (d, 3H; H-6.sup.IV), 1.17 ppm (d, 3H; H-6.sup.III); .sup.13C NMR (126 MHz, CDCl.sub.3): δ 174.1, 137.5, 137.5, 137.3, 137.3, 129-128, 100.6, 100.5, 100.4, 98.7, 77.8, 77.6, 77.1, 76.7, 74.1, 73.7, 73.4, 72.4, 72.3, 72.3, 72.2, 67.9, 67.9, 67.6, 67.5, 67.3, 67.2, 64.5, 64.4, 64.3, 64.0, 51.7, 34.1, 29.2, 25.8, 24.8, 18.8, 18.7, 18.7, 18.5 ppm; HRMS (ESI): m/z calcd for C.sub.59H.sub.74N.sub.12NaO.sub.15 [M+Na].sup.+: 1213.5289. found: 1213.5278.

5-Methoxycarbonylpentyl 2-O-acetyl-4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→3) 2-O-acetyl-4-azido-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (34)

(104) Donor 31 (104 mg, 70 μmol) and acceptor 33 (73 mg, 61 μmol) were dissolved in toluene (1.5 mL) with molecular sieves and TMSOTf (1 μL, 5 μmol) was added. The mixture was stirred 3 hours at room temperature before being quenched with one drop of pyridine. After filtration, a purification on silica gel column using toluene with a gradient of ethyl acetate (from 0% to 10%) gave pure nonasaccharide 34 (45 mg, 30%): [α].sub.D.sup.20=+95 (c=1.0 in CHCl.sub.3); .sup.1H NMR (600 MHz, CDCl.sub.3): δ 7.4-7.2 (m, 40H; H—Ar), 5.40 (dd, .sup.3J.sub.1,2=1.9 Hz, .sup.3J.sub.2,3=3.1 Hz, 1H; H-2.sup.IX), 5.12 (dd, .sup.3J.sub.1,2=1.9 Hz, .sup.3J.sub.2,3=3.2 Hz, 1H; H-2.sup.V), 4.97 (d, .sup.3J.sub.1,2=1.5 Hz, 2H; H-1), 4.94 (d, .sup.3J.sub.1,2=1.8 Hz, 1H; H-1), 4.93 (d, .sup.3J.sub.1,2=1.8 Hz, 1H; H-1), 4.86 (d, .sup.3J.sub.1,2=1.7 Hz, 1H; H-1), 4.85 (d, .sup.3J.sub.1,2=1.8 Hz, 1H; H-1), 4.84 (d, .sup.3J.sub.1,2=1.7 Hz, 1H; H-1.sup.IX), 4.82 (d, .sup.3J.sub.1,2=1.5 Hz, 1H; H-1.sup.V), 4.59 (d, .sup.3J.sub.1,2=1.1 Hz, 1H; H-1.sup.I), 3.95 (dd, .sup.3J.sub.2,3=2.8 Hz, 1H; H-2), 3.87 (dd, .sup.3J.sub.2,3=3 Hz, 1H; H-2), 3.85 (dd, .sup.3J.sub.2,3=2.8 Hz, 1H; H-2), 3.84 (dd, .sup.3J.sub.2,3=3 Hz, 1H; H-2), 3.83 (dd, .sup.3J.sub.2,3=3 Hz, 1H; H-2), 3.80 (dd, .sup.3J.sub.2,3=3 Hz, 1H; H-2), 3.76 (dd, .sup.3J.sub.2,3=3 Hz, 1H; H-2.sup.I), 3.57 (dt, .sup.2J=9.7 Hz, .sup.3J.sub.a,b=6.7 Hz, 1H; H-a), 3.32 (dt, .sup.3J.sub.a,b=6.4 Hz, 1H; H-a), 2.32 (t, .sup.3J.sub.d,e=7.5 Hz, 2H; H-e), 2.10, 2.06 (2s, 6H; Ac), 1.64 (quint, .sup.3J.sub.c,d=7.6 Hz, 2H; H-d), 1.55 (m, 2H; H-b), 1.33 (m, 2H; H-c), 1.27-1.12 ppm (9d, .sup.3J.sub.5,6=6.2 Hz, 27H; H-6); .sup.13C NMR (126 MHz, CDCl.sub.3): δ 174.1 (C-f), 169.9, 169.6, 137.6, 137.5, 137.5, 137.4, 137.4, 137.3, 137.3, 137.3, 129-128, 100.8, 100.6, 100.5, 100.3, 100.2, 100.1, 99.2, 98.8, 98.7, 77.6, 77.0, 76.8, 76.8, 76.7, 76.6, 76.6, 76.0, 75.6, 74.3, 74.2, 73.7, 73.6, 73.6, 73.5, 73.4, 72.4, 72.4, 72.4, 72.3, 72.2, 72.2, 72.1, 71.7, 70.6, 68.4, 68.2, 68.0, 68.0, 68.0, 67.9, 67.8, 67.8, 67.3, 67.7, 67.2, 64.6, 64.5, 64.4, 64.4, 64.4, 64.2, 64.2, 64.2, 64.0, 51.7, 34.1, 29.2, 25.8, 24.8, 21.1, 21.0, 18.8, 18.7, 18.7, 18.7, 18.6, 18.6, 18.5, 18.5, 18.4 ppm; HRMS (ESI): m/z calcd for C.sub.121H.sub.147N.sub.27NaO.sub.32 [M+Na].sup.+: 2513.0598. found: 2513.0561.

5-Methoxycarbonylpentyl 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→3) 4-azido-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (35)

(105) Nonaccharide 34 (45 mg, 18 mol) in methanol (2 mL) was treated with a 0.1 M solution of sodium methoxide (0.1 mL). The reaction was stirred 2 hours at room temperature and then neutralized with ion exchange resin H.sup.+. Filtration, evaporation and chromatography on silica gel (eluent: ethyl acetate/hexane 1:6) gave pure compound 35 (34 mg, 78%): [α].sub.D.sup.20=+100 (c=1.0 in CHCl.sub.3); .sup.1H NMR (600 MHz, CDCl.sub.3): δ 7.4-7.3 (m, 40H; H—Ar), 4.99 (d, .sup.3J.sub.1,2=1.6 Hz, 1H; H-1), 4.97 (d, .sup.3J.sub.1,2=1.6 Hz, 1H; H-1), 4.96 (d, .sup.3J.sub.1,2=1.6 Hz, 1H; H-1), 4.94 (br s, 2H; H-1), 4.87 (d, .sup.3J.sub.1,2=1.6 Hz, 1H; H-1), 4.87 (d, .sup.3J.sub.1,2=1.6 Hz, 1H; H-1), 4.86 (d, .sup.3J.sub.1,2=1.7 Hz, 1H; H-1), 4.59 (d, .sup.3J.sub.1,2=1.6 Hz, 1H; H-1.sup.I), 3.99 (m, 2H; H-2), 3.93 (m, 2H; H-2), 3.91 (br s, 1H; H-2), 3.85 (dd, .sup.3J.sub.1,2=1.6 Hz, .sup.3J.sub.2,3 3 Hz, 1H; H-2), 3.83 (dd, .sup.3J.sub.1,2=1.6 Hz, .sup.3J.sub.2,3=3 Hz, 1H; H-2), 3.81 (dd, .sup.3J.sub.2,3=3 Hz, 1H; H-2), 3.77 (dd, .sup.3J.sub.2,3=3 Hz, 1H; H-2.sup.I), 3.68 (s, 3H; H-g), 3.57 (dt, .sup.2J=9.7 Hz, .sup.3J.sub.a,b=6.7 Hz, 1H; H-a), 3.32 (dt, .sup.3J.sub.a,b=6.4 Hz, 1H; H-a), 2.32 (t, .sup.3J.sub.d,e=7.5 Hz, 2H; H-e), 1.64 (quint, .sup.3J.sub.c,d=7.6 Hz, 2H; H-d), 1.55 (m, 2H; H-b), 1.33 (m, 2H; H-c), 1.27, 1.27, 1.26, 1.25, 1.20, 1.20, 1.19, 1.15, 1.15 ppm (9d, .sup.3J.sub.5,6=6.2 Hz, 27H; H-6); .sup.13C NMR (126 MHz, CDCl.sub.3): δ 174.1, 137.5, 137.5, 137.5, 137.3, 137.3, 137.3, 137.3, 137.3, 129-128, 100.8, 100.7, 100.6, 100.6, 100.5, 100.4, 100.3, 100.2, 98.7, 79.0, 77.8, 77.6, 77.5, 77.4, 77.0, 76.8, 76.7, 74.2, 73.6, 73.6, 73.5, 73.4, 73.4, 73.3, 72.4, 72.4, 72.4, 72.4, 72.4, 72.3, 72.2, 72.2, 69.6, 68.5, 68.2, 68.0, 68.0, 68.0, 67.9, 67.6, 67.5, 67.3 (C-5), 67.2 (C-2), 67.7 (C-a), 64.5, 64.4, 64.4, 64.4, 64.3, 64.3, 64.1, 64.0, 64.0, 51.7, 34.1, 29.2, 25.8, 24.8, 18.8, 18.7, 18.7, 18.7, 18.6, 18.6, 18.6, 18.5, 18.4 ppm; HRMS (ESI): m/z calcd for C.sub.117H.sub.143N.sub.27NaO.sub.30 [M+Na].sup.+: 2429.0386. found: 2429.0353.

5-Methoxycarbonylpentyl 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→3) 4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranoside (2)

(106) A solution of nonasaccharide 35 (34 mg, 14 mol) in a mixture pyridine/NEt.sub.3 1:1 (4 mL) was saturated with H.sub.2S for 1 h and the media was then stirred for 24 h at room temperature. The solvent was co-evaporated with toluene and mass spectrometry of the crude product showed only one peak corresponding to compound 36 and no more product from incomplete reduction: HRMS (ESI): m/z calcd for C.sub.117H.sub.162N.sub.9O.sub.30 [M+H].sup.+: 2173.1439. found: 2173.1422. This crude material was directly used for formylation.

(107) Compound 36 was dissolved in methanol (3 mL) and a solution of acetic anhydride/formic acid 2:1 (0.3 mL) was added. The mixture was stirred at room temperature overnight. The solvent was evaporated and nonasaccharide 37 (21 mg, 62% over 2 steps) was purified with a chromatography column (eluent: MeOH/DCM 1:10): HRMS (ESI): m/z calcd for C.sub.126H.sub.161N.sub.9Na.sub.2O.sub.39 [M+2Na].sup.2+: 1235.0338. found: 1235.0333.

(108) Compound 37 (21 mg, 8.6 μmol) in solution in acetic acid (5 mL) with palladium on charcoal was stirred overnight at room temperature under atmosphere of hydrogen. After filtration and concentration, nonasaccharide 2 (7 mg, 48%) was purified on a reverse phase HPLC column (MeCN/H.sub.2O 18:82): .sup.1H NMR (600 MHz, D.sub.2O): δ 8.21-8.00 (m, 9H; NCHO), 5.21-4.84 (m, 9H; 9×H-1), 4.20-3.78 (m, 34H; 9×H-2, 9×H-3, 7×H-4, 9×H-5), 3.74-3.68 (m, 4H; H-a, H-g), 3.56-3.3.34 (m, 3H; 2×H-4, H-a), 2.40 (t, .sup.3J.sub.d,e=7.4 Hz, 2H; H-e), 1.66-1.58 (m, 4H; H-b, H-d), 1.42-1.33 (m, 2H; H-c), 1.30-1.15 ppm (m, 27H; 9×H-6); HRMS (ESI): m/z calcd for C.sub.70H.sub.113N.sub.9O.sub.39Na.sub.2 [M+2Na].sup.2+: 874.846. found: 874.8467.

(109) Proton labeling of the linker for compounds 38-41 is as follows:

(110) ##STR00024##

(2-Aminoethylamido)carbonylpentyl 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→3) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranoside (38)

(111) A solution of 1 (12 mg, 11.8 mol) in freshly distilled 1,2-diaminoethane (550 μL) was stirred at 50° C. for 48 h then concentrated. The residue was dissolved in water, neutralized with acetic acid and purified by reversed phase HPLC on C18 column in gradient water-acetonitrile to give 38 as a white powder (10 mg, 81%): .sup.1H NMR (500 MHz, D.sub.2O): δ 8.21-8.19 and 8.02-8.00 (m, 5H; NCHO), 5.17-4.86 (m, 5H; 5×H-1), 4.19-3.76 (m, 19H; 5×H-2, 5×H-3, 4×H-4, 5×H-5), 3.71-3.66 (m, 1H; H-a), 3.55-3.51 (m, 1H; H-a), 3.50-3.32 (m, 1H; H-4), 3.27 (t, .sup.3J.sub.f,g=6.2 Hz, 2H; H-f), 2.80 (t, 2H; H-g), 2.26 (t, .sup.3J.sub.d,e=7.4 Hz, 2H; H-e), 1.64-1.54 (m, 4H; H-b, H-d), 1.40-1.31 ppm (m, 2H; H-c), 1.30-1.16 (m, 15H; 5×H-6); HRMS (ESI): m/z calcd for C.sub.43H.sub.74N.sub.7O.sub.22 [M+H].sup.+: 1040.4881. found: 1040.4879.

1-[(2-Aminoethylamido)carbonylpentyl 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→3) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranoside]-2-butoxycyclobutene-3,4-dione (40)

(112) To a solution of amine 38 (5 mg, 4.8 mol) in a mixture of water (300 μL) and ethanol (200 μL) a solution of 3,4-dibutoxy-3-cyclobutene-1,2-dione (20% in ethanol, 26 μL) was added and pH was adjusted to 8 by addition of sodium bicarbonate solution. After 0.5 h, when TLC indicated the reaction was complete, the reaction mixture was neutralized with acetic acid and purified by reversed phase HPLC (C18) using gradient water-acetonitrile. Product which came out at 20% of acetonitrile was lyophilized to afford squarate 40 as a white powder (4.2 mg, 73%): .sup.1H NMR (600 MHz, D.sub.2O): δ 8.23-8.18 and 8.06-7.98 (m, 5H; NCHO), 5.18-5.87 (m, 5H; 5×H-1), 4.69 (m, 2H; H-h), 3.72-3.58 (m, 3H; H-a, 2×H-f), 3.51-3.36 (m, 4H; H-4, H-a, 2×H-g). 2.24-2.17 (m, 2H; H-e), 1.82-1.74 (m, 2H; H-i), 1.61-1.49 (m, 4H; H-b, H-d), 1.49-1.38 (m, 2H; H-j), 1.33-1.18 (m, 17H; 15×H-6, H-c), 0.96-0.91 (m, 3H; H-k); HRMS (ESI): m/z calcd for C.sub.51H.sub.81N.sub.7NaO.sub.25 [M+Na].sup.+: 1214.5174. found: 1214.5177.

(2-Aminoethylamido)carbonylpentyl 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→3) 4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranoside (39)

(113) Methyl ester 2 (5.9 mg, 3.46 μmol) in freshly distilled ethylenediamine (400 μL) was stirred at 50° C. for 48 h. TLC indicated the reaction was almost complete. The mixture was concentrated, dissolved in water and neutralized with 10% acetic acid. It was first purified on a SepPak C18 cartridge washing it first with water and then with methanol. Methanol fractions containing the product were combined and concentrated and then purified on HPLC (C18) using water-acetonitrile gradient. The product came out at 12% of acetonitrile. It was concentrated and lyophilized to yield the title amine 39 as a white powder (4.5 mg, 75%): .sup.1H NMR (500 MHz, D.sub.2O):δ 8.22-7.99 (m, 9H; NCHO), 5.20-4.86 (m, 9H; 9×H-1), 4.18-3.76 (m, 34H; 9×H-2, 9×H-3, 7×H-4, 9×H-5), 3.72-3.66 (m, 1H; H-a), 3.55-3.29 (m, 5H; 2×H-4, H-a, H-f), 2.81 (m, 2H; H-g), 2.26 (t, .sup.3J.sub.d,e=7.4 Hz, 2H; H-e), 1.65-1.58 (m, 4H; H-b, H-d), 1.40-1.30 (m, 2H; H-c), 1.30-1.16 (m, 27H; 9×H-6); HRMS (ESI): m/z calcd for C.sub.71H.sub.118N.sub.11O.sub.38 [M+H].sup.+: 1732.7634. found: 1732.7596.

1-[(2-Aminoethylamido)carbonylpentyl 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→3) 4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranoside]-2-butoxycyclobutene-3,4-dione (41)

(114) The amine 39 (4.5 mg, 2.5 μmol) was dissolved in water (0.3 mL) and ethanol (0.2 mL) was added to the solution. It was stirred at room temperature and a solution of 3,4-dibutoxy-3-cyclobutene-1,2-dione in ethanol (20%, 17 μL) was added and the pH of the reaction mixture was adjusted to 8 by careful addition of NaHCO.sub.3 solution. When TLC indicated the reaction was complete the mixture was neutralized with 10% acetic acid and purified by reversed phase HPLC (C18) using gradient water-acetonitrile. The product which came out at 20% of acetonitrile was concentrated and lyophilized to afford title compound 41 as a white powder (3.7 mg, 76%): .sup.1H NMR (500 MHz, D.sub.2O): δ 8.31-8.08 (m, 9H; NCHO), 5.30-4.94 (m, 9H; 9×H-1), 4.82-4.74 (m, 2H; H-h), 4.28-3.42 (m, 42H; 9×H-2, 9×H-3, 9×H-4, 9×H-5, H-a, H-f, H-g), 2.33-2.25 (m, 2H; H-e), 1.91-1.82 (m, 2H; H-i), 1.70-1.57 (m, 4H; H-b, H-d), 1.56-1.48 (m, 2H; H-j), 1.42-1.23 (m, 29H; 9×H-6, H-c), 1.05-0.99 (m, 3H; H-k); HRMS (ESI): m/z calcd for C.sub.79H.sub.125N.sub.11Na.sub.2O.sub.41 [M+2Na].sup.+2: 964.8909. found: 964.8904.

Methyl 4-azido-2,3-di-O-benzoyl-4,6-dideoxy-α-D-mannopyranoside (50) (Eichler et al. (1991) Glycoconjugate 8, 69-74)

(115) Benzoyl chloride (2.0 mL, 17.2 mmol) was added dropwise to a stirred solution of Methyl 4-azido-4,6-dideoxy-α-D-mannopyranoside 3 (Bundle et al. (1998) Carbohydr. Res. 174, 239-251) (1.59 g, 7.82 mmol) in pyridine (5 mL) containing DMAP (0.191 g, 1.56 mmol) at 0° C. The resulting mixture was stirred under argon for 10 h at room temperature. Then CH.sub.3OH (2 mL) was added to the reaction mixture. It was stirred for 10 min, then diluted with CH.sub.2Cl.sub.2 (˜100 mL) and washed with aq. HCl (1M, 2×50 mL), water (100 mL), 5% aq. NaHCO.sub.3 (50 mL), and brine (30 mL). The organic phase was separated, dried over MgSO.sub.4, and concentrated in vacuo. The residue was purified by column chromatography on silica gel (ethyl acetate-hexane gradient elution) to afford the title compound (3.12 g, 97%) as a white foam. Analytical data for 50: Rf=0.40 (ethyl acetate/hexane, 1/9, v/v); [α].sub.D.sup.21=−130.8 (c=1.1, CHCl.sub.3); .sup.1H NMR (600 MHz, CDCl.sub.3): δ 8.21-7.31 (m, 10H, H—Ar), 5.60 (dd, 1H, J.sub.2,3=1.8 Hz, H-2), 5.59 (dd, 1H, J.sub.3,4=3.6 Hz, H-3), 4.85 (d, 1H, J.sub.1,2=1.2 Hz, H-1), 3.83 (dq, 1H, J.sub.4,5=10.2 Hz, J.sub.5,6=6.0 Hz, H-5), 3.76 (dd, 1H, J.sub.4,5=10.2 Hz, H-4), 3.45 (s, 3H, —OCH.sub.3), 1.48 ppm (d, 3H, J.sub.5,6=6.0 Hz, H-6); .sup.13C NMR (126 MHz, CDCl.sub.3): δ 165.4, 165.3, 133.8, 133.5, 133.3, 130.2, 129.8, 129.8, 129.5, 129.3, 128.6, 128.5, 128.4, 98.6, 71.1, 69.8, 66.9, 63.5, 55.4, 18.6 ppm; HRMS (ESI): m/z calcd for C.sub.21H.sub.21N.sub.3O.sub.6Na [M+Na].sup.+: 434.1323. found: 434.1317.

1-O-Acetyl-4-azido-2,3-di-O-benzoyl-4,6-dideoxy-α-D-mannopyranose (51)

(116) A solution of 50 (3.10 g, 7.54 mmol) in acetic anhydride/acetic acid/sulfuric acid (50:20:0.5, 70 mL) was stirred at room temperature for 6 h, and then poured into ice-cold 1M K.sub.2CO.sub.3 solution (100 mL). The mixture was then diluted with CH.sub.2Cl.sub.2 (˜200 mL) and washed with water (2×100 mL), 5% aq. NaHCO.sub.3 (50 mL), and brine (30 mL). The organic phase was separated, dried over MgSO.sub.4, and concentrated in vacuo. The residue was purified by column chromatography on silica gel (ethyl acetate-hexane gradient elution) to afford the title compound (3.04 g, 92%) as a white foam. Analytical data for 51: Rf=0.30 (ethyl acetate/hexane, 1/9, v/v); [α].sub.D.sup.21=−119.8 (c=1.1, CHCl.sub.3); .sup.1H NMR (500 MHz, CDCl.sub.3): δ 7.35-8.09 (m, 10H, H—Ar), 6.25 (d, J=1.5 Hz, 1H, H-1), 5.65 (dd, J.sub.2,3=3.5 Hz, 1H, H-2), 5.61 (dd, J.sub.3,4=10.1 Hz, 1H, H-3), 3.91 (dq, J=10.2, 6.2 Hz, 1H, H-5), 3.85 (dd, J.sub.4,5=10.1 Hz, 1H, H-4), 2.24 (s, 3H, —OC—CH.sub.3), 1.51 (d, J.sub.5,6=6.2 Hz, 3H, H-6); .sup.13C NMR (126 MHz, CDCl.sub.3): δ 168.4, 165.4, 165.1, 133.7, 133.5, 129.9, 129.8, 129.1, 129.0, 128.6, 128.5, 90.7, 70.9, 69.4, 68.6, 63.0, 21.0, 18.7 ppm; HRMS (ESI): m/z calcd for C.sub.22H.sub.21N.sub.3O.sub.7Na [M+Na].sup.+: 462.1272. found: 462.1265.

4-Azido-2,3-di-O-benzoyl-4,6-dideoxy-α/β-D-mannopyranose (52)

(117) Hydrazine acetate (0.056 g, 0.612 mmol) was added to a stirred solution of 51 (0.224 g, 0.510 mmol) in DMF (1 mL) under argon atmosphere and the solution was stirred at 60° C. for 30 min. Then the mixture was cooled to room temperature, diluted with ethyl acetate (50 mL), washed with water (2×50 mL) and brine (30 mL). The organic phase was separated, dried over MgSO.sub.4, and concentrated in vacuo. The residue was purified by column chromatography on silica gel (ethyl acetate-hexane gradient elution) to afford the title compound (0.191 g, 95%) as a white foam. Analytical data for 52: Rf=0.30 (ethyl acetate/hexane, 1/4, v/v); .sup.1H NMR (500 MHz, CDCl.sub.3): α:β ratio=7:1; δ 7.11-8.06 (m, 20H, H—Ar), 5.78 (dd, J.sub.2,3=3.0 Hz, 1H, H-2.sub.β), 5.66 (dd, J.sub.3,4=10 Hz, 1H, H-3.sub.α), 5.63 (dd, J.sub.2,3=3.2 Hz, 1H, H-2.sub.α), 5.36 (dd, J.sub.1,2=1.6 Hz, 1H, H-1.sub.α), 5.30 (dd, J.sub.3,4=10.3 Hz, 1H, H-3.sub.β), 5.07 (dd, J.sub.1,2=1.5 Hz, 1H, H-1.sub.β), 1.52 (d, J.sub.5,6=6.0 Hz, 3H, H-6.sub.β), 1.45 ppm (d, J.sub.5,6=6.2 Hz, 3H, H-6.sub.α); .sup.13C NMR (126 MHz, CDCl.sub.3): δ 166.1, 165.5, 165.4, 133.8, 133.5, 133.4, 130.0, 129.8 (×2), 129.4, 129.2, 129.0 (×2), 128.9, 128.7, 128.6, 128.5, 128.4, 128.2, 92.9, 92.1, 73.3, 71.4, 70.9, 70.8, 70.2, 67.1, 63.6, 62.8, 18.7, 18.6 ppm; HRMS (ESI): m/z calcd for C.sub.20H.sub.19N.sub.3O.sub.6Na [M+Na].sup.+: 420.1166. found: 420.1163.

4-Azido-2,3-di-O-benzoyl-4,6-dideoxy-α-D-mannopyranosyl trichloroacetimidate (53)

(118) To a stirred solution of 52 (2.0 g, 5.03 mmol) in CH2Cl2 (20 mL) containing CCl3CN (10.1 mL, 100 mmol), DBU (0.150 mL, 1.00 mmol) was added at room temperature under an argon atmosphere. After 10 min, solvents were evaporated in vacuo and the residue was purified by column chromatography on silica gel (ethyl acetate hexane gradient elution) to afford the title compound (2.335 g, 86%) as an off-white foam. Analytical data for 53: Rf=0.60 (ethyl acetate/hexane, 1/4, v/v); [α].sub.D.sup.21=−101.9 (c=1.0, CHCl.sub.3); .sup.1H NMR (500 MHz, CDCl.sub.3): δ 8.79 (s, 1H, N—H), 7.33-8.09 (m, 10H, H—Ar), 6.43 (d, J.sub.1,2=1.5 Hz, 1H, H-1), 5.85 (dd, J.sub.2,3=3.5 Hz, 1H, H-2), 5.68 (dd, J.sub.3,4=10.5 Hz, 1H, H-3), 4.07 (dq, J=10.2, 6.2 Hz, 1H, H-5), 3.88 (dd, J.sub.4,5=10.2 Hz, 1H, H-4), 1.53 ppm (d, J.sub.5,6=6.2 Hz, 3H, H-6); .sup.13C NMR (126 MHz, CDCl.sub.3): δ 165.3, 165.1, 160.0, 133.7, 133.5, 129.9, 129.8, 129.1, 129.0, 128.7, 128.5, 94.7, 90.7, 70.9, 69.9, 68.1, 62.9, 18.7 ppm; HRMS (ESI): m/z calcd for C.sub.22H.sub.19Cl.sub.3N.sub.4O.sub.6Na [M+Na].sup.+: 563.0262. found: 563.0251.

Methyl 4-azido-3-O-benzoyl-2-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (55)

(119) Benzoyl chloride (0.872 mL, 7.5 mmol) was added dropwise to a stirred solution of 54 (Peters & Bundle (1989) Can. J. Chem. 67, 497-502) (2.0 g, 6.82 mmol) in pyridine (10 mL) containing DMAP (0.166 g, 1.36 mmol) at 0° C. The resulting mixture was stirred under argon for 3 h at room temperature. Then CH.sub.3OH (2 mL) was added to the reaction mixture which was stirred for 10 min, then diluted with CH.sub.2Cl.sub.2 (˜80 mL) and washed with aq. HCl (1M, 2×50 mL), water (100 mL), 5% aq. NaHCO.sub.3 (50 mL), and brine (30 mL). The organic phase was separated, dried over MgSO.sub.4, and concentrated in vacuo. The residue was purified by column chromatography on silica gel (ethyl acetate-hexane gradient elution) to afford the title compound (2.47 g, 91%) as oil. Analytical data for 55: Rf=0.50 (ethyl acetate/hexane, 1/9, v/v); [α].sub.D.sup.21=−18.8 (c=1.1, CHCl.sub.3); .sup.1H NMR (600 MHz, CDCl.sub.3): δ 7.13-8.10 (m, 10H, H—Ar), 5.32 (dd, J.sub.3,4=10.4 Hz, 1H, H-3), 4.71 (d, J.sub.1,2=1.4 Hz, 1H, H-1), 3.37 (s, 3H, —OCH.sub.3), 1.42 ppm (d, J.sub.5,6=6.2 Hz, 3H, H-6); .sup.13C NMR (126 MHz, CDCl.sub.3): δ 165.5, 137.5, 133.3, 129.9, 129.5, 128.5, 128.3, 127.8 (×2), 98.9, 74.8, 73.3, 73.2, 67.0, 63.2, 55.0, 18.5 ppm; HRMS (ESI): m/z calcd for C.sub.21H.sub.23N.sub.3O.sub.5Na [M+Na].sup.+: 420.1530. found: 420.1521.

1-O-Acetyl-4-azido-3-O-benzoyl-2-O-benzyl-4,6-dideoxy-α-D-mannopyranose (56)

(120) A solution of 55 (1.40 g, 3.52 mmol) in acetic anhydride/acetic acid/sulfuric acid (50:20:0.5, 35 mL) was stirred at room temperature for 3 h, and then poured into ice-cold 1M K.sub.2CO.sub.3 solution (100 mL). The mixture was then diluted with CH.sub.2Cl.sub.2 (˜100 mL) and washed with water (2×80 mL), 5% aq. NaHCO.sub.3 (50 mL), and brine (30 mL). The organic phase was separated, dried over MgSO.sub.4, and concentrated in vacuo. The residue was purified by column chromatography on silica gel (ethyl acetate-hexane gradient elution) to afford the title compound (1.22 g, 81%) as a white foam. Analytical data for 56: Rf=0.30 (ethyl acetate/hexane, 1/9, v/v); [α].sub.D.sup.21=−9.9 (c=1.5, CHCl.sub.3); .sup.1H NMR (500 MHz, CDCl.sub.3): δ 7.10-8.08 (m, 10H, H—Ar), 6.17 (d, J.sub.1,2=1.3 Hz, 1H, H-1), 5.29 (dd, J.sub.3,4=10.5 Hz, 1H, H-3), 4.00 (dd, J.sub.2,3=3.5 Hz, 1H, H-2), 2.15 (s, 3H, —OC—CH.sub.3), 1.42 ppm (d, J.sub.5,6=6.1 Hz, 3H, H-6); .sup.13C NMR (126 MHz, CDCl.sub.3): δ 169.0, 165.6, 136.9, 133.5, 129.9, 129.2, 128.5, 128.4, 128.0, 127.9, 91.1, 73.5, 73.0, 72.7, 69.6, 62.6, 21.0, 18.5 ppm; HRMS (ESI): m/z calcd for C.sub.22H.sub.23N.sub.3O.sub.6Na [M+Na].sup.+: 448.1479. found: 448.1471.

4-Azido-3-O-benzoyl-2-O-benzyl-4,6-dideoxy-α/β-D-mannopyranose (57)

(121) Hydrazine acetate (0.310 g, 3.35 mmol) was added to a stirred solution of 56 (1.190 g, 2.79 mmol) in DMF (10 mL) under argon atmosphere and stirred at 60° C. for 30 min. Then the mixture was cooled to room temperature, diluted with ethyl acetate (100 mL), washed with water (2×80 mL) and brine (30 mL). The organic phase was separated, dried over MgSO.sub.4, and concentrated in vacuo. The residue was purified by column chromatography on silica gel (ethyl acetate-hexane gradient elution) to afford the title compound (0.912 g, 85%) as a white foam. Analytical data for 57: Rf=0.30 (ethyl acetate/hexane, 1/4, v/v); .sup.1H NMR (500 MHz, CDCl.sub.3): α:β ratio=5:1; δ 7.16-8.14 (m, 20H, H—Ar), 5.43 (dd, J.sub.3,4=10.4 Hz, 1H, H-3.sub.α), 5.27 (dd, J.sub.1,2=1.8 Hz, J.sub.1,—OH=3.3 Hz, 1H, H-1.sub.α), 5.13 (dd, J.sub.3,4=10.5 Hz, 1H, H-3.sub.β), 4.82 (dd, J.sub.1,2=1.5 Hz, J.sub.1,—OH=11.6 Hz, 1H, H-1.sub.β), 4.14 (dd, J.sub.2,3=3.0 Hz, 1H, H-2.sub.β), 4.06 (dd, J.sub.2,3=3.1 Hz, 1H, H-2.sub.α), 1.46 (d, J.sub.5,6=6.2 Hz, 3H, H-6.sub.β), 1.42 ppm (d, J.sub.5,6=6.1 Hz, 3H, H-6.sub.α); .sup.13C NMR (126 MHz, CDCl.sub.3): δ 165.6 (×2), 137.4, 137.1, 133.8, 133.4, 129.9, 129.5, 128.9, 128.7, 128.5, 128.3 (×2), 128.2, 127.9, 127.8, 93.2, 92.7, 76.3, 75.8, 75.7, 75.0, 73.3, 72.9, 70.9, 67.2, 63.2, 62.6, 18.6, 18.5 ppm; HRMS (ESI): m/z calcd for C.sub.20H.sub.21N.sub.3O.sub.5Na [M+Na].sup.+: 406.1373. found: 406.1366.

4-Azido-3-O-benzoyl-2-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl trichloroacetimidate (58)

(122) To a stirred solution of 57 (0.890 g, 2.32 mmol) in CH2Cl2 (15 mL) containing CCl3CN (4.65 mL, 46.4 mmol), DBU (70 μL, 0.464 mmol) was added at room temperature under argon atmosphere. After 10 min, solvents were evaporated in vacuo and the residue was purified by column chromatography on silica gel (ethyl acetate hexane gradient elution) to afford the title compound (1.136 g, 93%) as an off-white foam. Analytical data for 58: Rf=0.80 (ethyl acetate/toluene, 1/9, v/v); [α].sub.D.sup.21=−7.3 (c=1.5, CHCl.sub.3); .sup.1H NMR (600 MHz, CDCl.sub.3): δ 8.66 (s, 1H, —C═NH), 7.12-8.10 (m, 10H, H—Ar), 6.36 (d, J.sub.1,2=1.8 Hz, 1H, H-1), 5.39 (dd, J.sub.3,4=7.2 Hz, 1H, H-3), 4.26 (dd, J.sub.2,3=3.1 Hz, 1H, H-2), 3.91-3.99 (m, 2H, H-4, H-5), 1.48 ppm (d, J.sub.5,6=6.0 Hz, 3H, H-6); .sup.13C NMR (126 MHz, CDCl.sub.3): δ 165.5, 160.4, 137.0, 133.5, 129.9, 129.2, 128.5, 128.4, 128.0 (×2), 127.9, 95.3, 90.8, 73.2, 73.0, 72.8, 70.1, 62.5, 18.6 ppm; HRMS (ESI): m/z calcd for C.sub.22H.sub.21Cl.sub.3N.sub.4O.sub.5Na [M+Na].sup.+: 549.0470. found: 549.0465.

Methyl 4-azido-2-O-benzoyl-3-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (59)

(123) Benzoyl chloride (0.872 mL, 7.5 mmol) was added dropwise to a stirred solution of 4 (2.0 g, 6.82 mmol) in pyridine (10 mL) containing DMAP (0.166 g, 1.36 mmol) at 0° C. The resulting mixture was stirred under argon for 3 h at room temperature. Then CH.sub.3OH (2 mL) was added to the reaction mixture, stirred for 10 min, then diluted with CH.sub.2Cl.sub.2 (˜80 mL) and washed with aq. HCl (1M, 2×50 mL), water (100 mL), 5% aq. NaHCO.sub.3 (50 mL), and brine (30 mL). The organic phase was separated, dried over MgSO.sub.4, and concentrated in vacuo. The residue was purified by column chromatography on silica gel (ethyl acetate-hexane gradient elution) to afford the title compound (2.32 g, 86%) as a white foam. Analytical data for 59: Rf=0.50 (ethyl acetate/hexane, 1/9, v/v); [α].sub.D.sup.21=−27.9 (c=1.7, CHCl.sub.3); .sup.1H NMR (500 MHz, CDCl.sub.3): δ 7.21-8.10 (m, 10H, H—Ar), 5.55 (dd, J.sub.2,3=3.2 Hz, 1H, H-2), 4.77 (d, J.sub.1,2=1.8 Hz, 1H, H-1), 3.91 (dd, J.sub.3,4=9.7 Hz, 1H, H-3), 3.37 (s, 3H, —OCH.sub.3), 1.38 ppm (d, J.sub.5,6=6.0 Hz, 3H, H-6); .sup.13C NMR (126 MHz, CDCl.sub.3): δ 165.6, 137.3, 133.3, 129.9, 129.7, 128.4, 128.3, 128.1, 127.8, 98.8, 76.1, 71.4, 67.8, 66.8, 64.3, 55.1, 18.7 ppm; HRMS (ESI): m/z calcd for C.sub.21H.sub.23N.sub.3O.sub.5Na [M+Na].sup.+: 420.1530. found: 420.1528.

1-O-Acetyl-4-azido-2-O-benzoyl-3-O-benzyl-4,6-dideoxy-α/β-D-mannopyranose (60)

(124) A solution of 59 (2.06 g, 5.19 mmol) in acetic anhydride/acetic acid/sulfuric acid (50:20:0.5, 35 mL) was stirred at room temperature for 3 h, and then poured into ice-cold 1M K.sub.2CO.sub.3 solution (100 mL). The mixture was then diluted with CH.sub.2Cl.sub.2 (˜100 mL) and washed with water (2×80 mL), 5% aq. NaHCO.sub.3 (50 mL), and brine (30 mL). The organic phase was separated, dried over MgSO.sub.4, and concentrated in vacuo. The residue was purified by column chromatography on silica gel (ethyl acetate-hexane gradient elution) to afford the title compound (1.95 g, 89%) as a white foam.]α:β ratio=9:1 (isolated yield); Analytical data for 60: α/β ratio=9:1 (isolated yield); α-anomer: Rf=0.45 (ethyl acetate/hexane, 1.5/8.5, v/v); [α].sub.D.sup.21=+1.8 (c=1.9, CHCl.sub.3); .sup.1H NMR (500 MHz, CDCl.sub.3): δ 7.27-8.09 (m, 10H, H—Ar), 6.16 (d, J.sub.1,2=2.0 Hz, 1H, H-1), 2.13 (s, 3H, —OC—CH.sub.3), 1.40 ppm (d, J.sub.5,6=6.0 Hz, 3H, H-6); .sup.13C NMR (126 MHz, CDCl.sub.3): δ 168.3, 165.3, 136.9, 133.5, 129.9, 129.3, 128.5, 128.4, 128.2, 128.0, 91.1, 75.8, 71.6, 69.3, 66.7, 63.8, 20.9, 18.7 ppm; HRMS (ESI): m/z calcd for C.sub.22H.sub.23N.sub.3O.sub.6Na [M+Na].sup.+: 448.1479. found: 448.1475; β-anomer: Rf=0.40 (ethyl acetate/hexane, 1.5/8.5, v/v); [c].sub.D.sup.21=−50.2 (c=1.2, CHCl.sub.3); .sup.1H NMR (500 MHz, CDCl.sub.3): δ 7.27-8.13 (m, 10H, H—Ar), 5.82 (dd, J.sub.2,3=3.5 Hz, 1H, H-2), 5.77 (d, J.sub.1,2=1.5 Hz, 1H, H-1), 2.03 (s, 3H, —OC—CH.sub.3),), 1.46 ppm (d, J.sub.5,6=6.0 Hz, 3H, H-6); .sup.13C NMR (126 MHz, CDCl.sub.3): δ 168.8, 165.8, 136.6, 133.4, 130.0, 129.5, 128.5 (×2), 128.3, 128.1, 91.2, 78.1, 72.1, 71.4, 66.8, 63.6, 20.8, 18.6 ppm; HRMS (ESI): m/z calcd for C.sub.22H.sub.23N.sub.3O.sub.6Na [M+Na].sup.+: 448.1479. found: 448.1474.

4-Azido-2-O-benzoyl-3-O-benzyl-4,6-dideoxy-α/β-D-mannopyranose (61)

(125) Hydrazine acetate (0.572 g, 6.20 mmol) was added to a stirred solution of 60 (2.20 g, 5.17 mmol) in DMF (7 mL) under argon atmosphere and stirred at 60° C. for 30 min. Then the mixture was cooled to room temperature, diluted with ethyl acetate (100 mL), washed with water (2×80 mL) and brine (30 mL). The organic phase was separated, dried over MgSO.sub.4, and concentrated in vacuo. The residue was purified by column chromatography on silica gel (ethyl acetate-hexane gradient elution) to afford the title compound (1.83 g, 93%) as a white foam. Analytical data for 61: Rf=0.30 (ethyl acetate/hexane, 1/4, v/v); .sup.1H NMR (500 MHz, CDCl.sub.3): α/β ratio=5:1; δ 7.25-8.15 (m, 20H, H—Ar), 5.74 (dd, J.sub.2,3=3.5 Hz, 1H, H-2.sub.β), 5.60 (dd, J.sub.2,3=3.1 Hz, 1H, H-2.sub.α), 5.32 (d, J.sub.1,2=1.3 Hz, 1H, H-1.sub.α), 4.89 (s, 1H, H-1.sub.β), 1.47 (d, J.sub.5,6=6.2 Hz, 3H, H-6.sub.β), 1.40 ppm (d, J.sub.5,6=6.1 Hz, 3H, H-6.sub.α); .sup.13C NMR (126 MHz, CDCl.sub.3): δ 166.3, 165.7, 137.2, 136.8, 133.6, 133.3, 130.0, 129.9, 129.6, 129.2, 128.5, 128.4 (×2), 128.3, 128.2, 128.0, 127.8, 93.2, 92.5, 78.5, 75.6, 71.5 (×2), 71.3, 69.2, 68.2, 67.1, 64.3, 63.7, 18.7 (×2) ppm; HRMS (ESI): m/z calcd for C.sub.20H.sub.21N.sub.3O.sub.5Na [M+Na].sup.+: 406.1373. found: 406.1374.

4-Azido-2-O-benzoyl-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl trichloroacetimidate (62)

(126) To a stirred solution of 61 (1.820 g, 4.75 mmol) in CH2Cl2 (20 mL) containing CCl3CN (9.50 mL, 95.0 mmol), DBU (140 μL, 0.95 mmol) was added at room temperature under argon atmosphere. After 10 min, solvents were evaporated in vacuo and the residue was purified by column chromatography on silica gel (ethyl acetate-hexane gradient elution) to afford the title compound (2.0 g, 80%) as an off-white foam. Analytical data for 62: Rf=0.60 (ethyl acetate/hexane, 1/4, v/v); [α].sub.D.sup.21=−10.8 (c=1.0, CHCl.sub.3); .sup.1H NMR (500 MHz, CDCl.sub.3): δ 8.70 (s, 1H, —C═NH), 7.28-8.12 (m, 10H, H—Ar), 6.31 (d, J.sub.1,2=1.8 Hz, 1H, H-1), 5.67 (dd, J.sub.2,3=3.5 Hz, 1H, H-2), 3.98 (dd, J.sub.3,4=10.0 Hz, 1H, H-3), 3.57-3.66 (dd, J.sub.4,5=10.0 Hz, 1H, H-4), 1.38-1.44 ppm (d, J.sub.5,6=6.5 Hz, 3H, H-6); .sup.13C NMR (126 MHz, CDCl.sub.3): δ 165.3, 159.8, 136.7, 133.5, 129.9, 129.3, 128.6 (×3), 128.5 (×2), 128.4, 128.1, 95.0, 90.7, 75.2, 71.6, 69.9, 66.4, 63.7, 18.7 ppm; HRMS (ESI): m/z calcd for C.sub.22H.sub.21Cl.sub.3N.sub.4O.sub.5Na [M+Na].sup.+: 549.0470. found: 549.0475.

Ethyl 4-azido-2,3-di-O-benzoyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-1-thio-α-D-mannopyranoside (63)

(127) A mixture of glycosyl donor 53 (1.010 g, 1.87 mmol), glycosyl acceptor 13 Peters & Bundle (1989) Can. J. Chem. 67, 491-496) (0.550 g, 1.70 mmol), and freshly activated molecular sieves (3 Å, 2.0 g) in CH.sub.2Cl.sub.2 (30 mL) was stirred under argon for 5 h at room temperature. TMSOTf (67 μL, 0.374 mmol) was added and the resulting mixture was stirred for an additional 60 min. Then Et.sub.3N (1 mL) was added, the solid was filtered off and the residue was rinsed with CH.sub.2Cl.sub.2 (3×20 mL). The combined filtrate (˜100 mL) was washed with 20% aq. NaHCO.sub.3 (50 mL), water (50 mL), and brine (30 mL). The organic phase was separated, dried over MgSO.sub.4, and concentrated in vacuo. The residue was purified by column chromatography on silica gel (ethyl acetate-toluene gradient elution) to afford the title compound (1.073 g, 90%) as a white foam. Analytical data for 63: Rf=0.70 (ethyl acetate/toluene, 1/9, v/v); [α].sub.D.sup.21=−18.4 (c=1.1, CHCl.sub.3); .sup.1H NMR (600 MHz, CDCl.sub.3): δ 7.12-8.03 (m, 15H, H—Ar), 5.70 (dd, J.sub.2,3=3.2 Hz, 1H, H-2.sup.B), 5.62 (dd, J.sub.3,4=10.3 Hz, 1H, H-3.sup.B), 5.24 (d, J.sub.1,2=1.5 Hz, 1H, H-1.sup.A), 4.98 (d, J.sub.1,2=1.5 Hz, 1H, H-1.sup.B), 2.54-2.67 (m, 2H, S—CH.sub.2—), 1.44 (d, J.sub.5,6=6.2 Hz, 3H, H-6.sup.B), 1.37 (d, J.sub.5,6=6.1 Hz, 3H, H-6.sup.A), 1.29 ppm (t, J=7.4 Hz, 3H, S—CH.sub.2—CH.sub.3); .sup.13C NMR (126 MHz, CDCl.sub.3): δ 165.2, 164.9, 137.4, 133.4, 133.3, 129.8 (×2), 129.5, 129.3, 128.5 (×2), 128.4, 128.1, 127.9, 99.4, 83.3, 78.4, 76.5, 72.4, 70.8, 69.5, 67.8, 67.6, 64.2, 63.5, 25.6, 18.6, 18.4, 14.9 ppm; HRMS (ESI): m/z calcd for C.sub.35H.sub.38N.sub.6O.sub.8SNa [M+Na].sup.+: 725.2364. found: 725.2350.

Ethyl 4-azido-2-O-benzoyl-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-1-thio-α-D-mannopyranoside (64)

(128) A mixture of glycosyl donor 62 (1.980 g, 3.76 mmol), glycosyl acceptor 13 (1.106 g, 3.42 mmol), and freshly activated molecular sieves (3 Å, 4.0 g) in CH.sub.2Cl.sub.2 (30 mL) was stirred under argon for 5 h at room temperature. TMSOTf (0.136 mL, 0.753 mmol) was added and the resulting mixture was stirred for additional 1 h. Then Et.sub.3N (1 mL) was added, the solid was filtered off and the residue was rinsed with CH.sub.2Cl.sub.2 (3×30 mL). The combined filtrate (˜120 mL) was washed with 20% aq. NaHCO.sub.3 (50 mL), water (50 mL), and brine (30 mL). The organic phase was separated, dried over MgSO.sub.4, and concentrated in vacuo. The residue was purified by column chromatography on silica gel (ethyl acetate-toluene gradient elution) to afford the title compound (2.010 g, 85%) as a white foam. Analytical data for 64: Rf=0.50 (ethyl acetate/hexane, 1/9, v/v); [α].sub.D.sup.21=+50.0 (c=1.4, CHCl.sub.3); .sup.1H NMR (500 MHz, CDCl.sub.3): δ 7.21-8.11 (m, 15H, H—Ar), 5.63 (dd, J.sub.2,3=2.5 Hz, 1H, H-2.sup.B), 5.22 (d, J.sub.1,2=1.0 Hz, 1H, H-1.sup.A), 4.95 (d, J.sub.1,2=1.7 Hz, 1H, H-1.sup.B), 2.50-2.69 (m, 2H, S—CH.sub.2—), 1.37 (d, J.sub.5,6=6.2 Hz, 3H, H-6.sup.B), 1.33 (d, J.sub.5,6=6.2 Hz, 3H, H-6.sup.A), 1.28 ppm (t, J=7.3 Hz, 3H, S—CH.sub.2—CH.sub.3); .sup.13C NMR (126 MHz, CDCl.sub.3): δ 165.3, 137.3, 137.1, 133.3, 129.9, 129.7, 128.6, 128.5, 128.4 (×2), 128.2, 128.1, 127.9, 99.6, 83.3, 78.1, 76.6, 75.3, 72.3, 71.4, 67.7, 67.6, 64.4, 64.1, 25.6, 18.7, 18.5, 14.9 ppm; HRMS (ESI): m/z calcd for C.sub.35H.sub.40N.sub.6O.sub.7SNa [M+Na].sup.+: 711.2571. found: 711.2570.

5′-Methoxycarbonylpentyl 4-azido-2,3-di-O-benzoyl-4,6-dideoxy-α-D-mannopyranosyl (1→3) 4-azido-2-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (67)

(129) A mixture of glycosyl donor 53 (0.292 g, 0.540 mmol), glycosyl acceptor 65 (Saksena et al. (2008) Carbohydr. Res. 343, 1693-1706) (0.200 g, 0.491 mmol) and freshly activated molecular sieves (3 Å, 0.6 g) in CH.sub.2Cl.sub.2 (8 mL) was stirred under argon for 5 h at room temperature. TMSOTf (20 μL, 0.108 mmol) was added and the resulting mixture was stirred for an additional hour. Then Et.sub.3N (1 mL) was added, the solid was filtered off and the residue was rinsed with CH.sub.2Cl.sub.2 (3×20 mL). The combined filtrate (80 mL) was washed with 20% aq. NaHCO.sub.3 (40 mL), water (30 mL), and brine (20 mL). The organic phase was separated, dried over MgSO.sub.4, and concentrated in vacuo. The residue was purified by column chromatography on silica gel (ethyl acetate-toluene gradient elution) to afford the title compound (0.374 g, 97%) as a white foam. Analytical data for 67: Rf=0.40 (ethyl acetate/toluene, 0.5/9.5, v/v); [α].sub.D.sup.21=−41.2 (c=1.6, CHCl.sub.3); .sup.1H NMR (600 MHz, CDCl.sub.3): δ 7.29-8.06 (m, 15H, H—Ar), 5.72 (dd, J.sub.2,3=3.3 Hz, 1H, H-2.sup.B), 5.66 (dd, J.sub.3,4=10.3 Hz, 1H, H-3.sup.B), 5.29 (d, J.sub.1,2=1.8 Hz, 1H, H-1.sup.B), 4.82 (d, J.sub.1,2=1.8 Hz, 1H, H-1.sup.A), 4.73 3.66 (s, 3H, —OCH.sub.3), 1.37 (d, J.sub.5,6=6.0 Hz, 3H, H-6.sup.B), 1.36 (d, J.sub.5,6=6.0 Hz, 3H, H-6.sup.A); .sup.13C NMR (126 MHz, CDCl.sub.3): δ 174.0, 165.2, 165.1, 137.6, 133.5, 133.3, 129.8, 129.4, 129.3, 128.6, 128.5, 128.4, 127.9, 127.6, 99.2, 97.0, 78.6, 72.5, 70.7, 70.0, 67.7, 67.6 (×2), 64.7, 63.4, 51.5, 33.9, 29.0, 25.7, 24.6, 18.6, 18.5 ppm; HRMS (ESI): m/z calcd for C.sub.40H.sub.46N.sub.6O.sub.11Na [M+Na].sup.+: 809.3117. found: 809.3107.

5′-Methoxycarbonylpentyl 4-azido-2,3-di-O-benzoyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→3) 4-azido-2-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (68)

(130) A mixture of glycosyl donor 63 (0.151 g, 0.216 mmol), glycosyl acceptor 65 (Saksena et al. (2008) Carbohydr. Res. 343, 1693-1706) (0.080 g, 0.196 mmol) and freshly activated molecular sieves (3 Δ, 0.5 g) in CH.sub.2Cl.sub.2 (4 mL) was stirred under argon for 5 h at room temperature. MeOTf (133 μL, 1.17 mmol) was added and stirring was continued for additional 48 h. Then Et.sub.3N (1 mL) was added, the solid was filtered off and the residue was rinsed with CH.sub.2Cl.sub.2 (3×20 mL). The combined filtrate (70 mL) was washed with 20% aq. NaHCO.sub.3 (40 mL), water (30 mL), and brine (20 mL). The organic phase was separated, dried over MgSO.sub.4, and concentrated in vacuo. The residue was purified by column chromatography on silica gel (ethyl acetate-toluene gradient elution) to afford the title compound (0.182 g, 89%) as a white foam. Analytical data for 68: Rf=0.40 (ethyl acetate/toluene, 0.5/9.5, v/v); [α].sub.D.sup.21=−27.7 (c=2.4, CHCl.sub.3); .sup.1H NMR (600 MHz, CDCl.sub.3): δ 7.06-8.01 (m, 20H, H—Ar), 5.71 (dd, J.sub.2,3=3.3 Hz, 1H, H-2.sup.C), 5.58 (dd, J.sub.3,4=10.4 Hz, 1H, H-3.sup.C), 5.07 (s, 2H, H-1.sup.B, H-1.sup.C), 4.78 (d, J.sub.1,2=1.5 Hz, 1H, H-1.sup.A), 1.46 (d, J.sub.5,6=6.2 Hz, 3H, H-6.sup.C), 1.34 (d, J.sub.5,6=6.5 Hz, 3H, H-6.sup.B), 1.32 ppm (d, J.sub.5,6=6.2 Hz, 3H, H-6.sup.A); .sup.13C NMR (126 MHz, CDCl.sub.3): δ 174.0, 165.2, 164.9, 137.6, 137.4, 133.4, 133.2, 129.8, 129.7, 129.5, 129.3, 128.5 (×2), 128.4, 128.2, 127.9, 127.8, 127.7, 127.6, 101.0, 99.2, 97.0, 78.2, 77.8, 73.4, 72.6, 72.1, 70.9, 69.3, 68.2, 67.7, 67.3, 64.9, 63.7, 63.4, 51.5, 33.9, 29.0, 25.7, 24.6, 18.6 (×2), 18.5 ppm; HRMS (ESI): m/z calcd for C.sub.53H.sub.61N.sub.9O.sub.14Na [M+Na].sup.+: 1070.4230. found: 1070.4233.

5′-Methoxycarbonylpentyl 4-azido-3-O-benzoyl-2-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (69)

(131) A mixture of glycosyl donor 53 (0.168 g, 0.318 mmol), glycosyl acceptor 66 Saksena et al. (2005) Tetrahedron: Asymmetry 16, 187-197) (0.118 g, 0.289 mmol) and freshly activated molecular sieves (3 Å, 0.350 g) in PhMe (3 mL) was stirred under argon for 2 h at room temperature. Then it was heated to 95° C. and TMSOTf (12 μL, 0.064 mmol) was added, and the mixture stirred for additional 1 h. Then Et.sub.3N (1 mL) was added, the solid was filtered off and the residue was rinsed with CH.sub.2Cl.sub.2 (3×30 mL). The combined filtrate (100 mL) was washed with 20% aq. NaHCO.sub.3 (50 mL), water (30 mL), and brine (30 mL). The organic phase was separated, dried over MgSO.sub.4, and concentrated in vacuo. The residue was purified by column chromatography on silica gel (ethyl acetate-toluene gradient elution) to afford the title compound (0.203 g, 91%) as a white foam. Analytical data for 69: Rf=0.40 (ethyl acetate/toluene, 0.5/9.5, v/v); [α].sub.D.sup.21=−4.4 (c=1.2, CHCl.sub.3); .sup.1H NMR (500 MHz, CDCl.sub.3): δ 7.00-8.06 (m, 15H, H—Ar), 5.30 (dd, J.sub.3,4=9.9 Hz, 1H, H-3.sup.B), 5.07 (d, J.sub.1,2=1.7 Hz, 1H, H-1.sup.B), 4.70 (d, J.sub.1,2=1.8 Hz, 1H, H-1.sup.A), 3.69 (s, 3H, —OCH.sub.3), 1.39 (d, J=5.9 Hz, 3H, H-6.sup.A), 1.34 ppm (d, J=5.7 Hz, 3H, H-6.sup.B); .sup.13C NMR (126 MHz, CDCl.sub.3): δ 174.0, 165.4, 137.4, 137.3, 133.2, 129.8, 129.5, 128.5, 128.4, 128.2, 128.1, 127.8, 127.6, 99.1, 98.8, 78.6, 74.4, 73.3, 72.8, 72.6, 72.5, 67.6, 67.4, 67.3, 64.3, 63.1, 51.5, 33.9, 29.1, 25.7, 24.7, 18.5 (×2) ppm; HRMS (ESI): m/z calcd for C.sub.40H.sub.48N.sub.6O.sub.10Na [M+Na].sup.+: 795.3324. found: 795.3314.

5′-Methoxycarbonylpentyl 4-azido-2-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (70)

(132) Sodium methoxide (˜0.5 mL, 0.5 M solution) was added to a solution of 69 (0.910 g, 1.178 mmol) in CH.sub.3OH (20 mL) until pH ˜9 and the resulting mixture was stirred under argon for 4 h at room temperature. Then the reaction mixture was neutralized with Amberlite IR 120 (H.sup.+) ion exchange resin, the resin was filtered off and rinsed successively with CH.sub.3OH. The combined filtrate was concentrated in vacuo and purified by column chromatography on silica gel (ethyl acetate-toluene gradient elution) to afford the title compound (0.755 g, 96%) as oil. Analytical data for 70: Rf=0.50 (ethyl acetate/toluene, 1/9, v/v); [α].sub.D.sup.21=+43.2 (c=1.2, CHCl.sub.3); .sup.1H NMR (500 MHz, CDCl.sub.3): δ 7.11-7.41 (m, 10H, H—Ar), 5.11 (d, J.sub.1,2=1.3 Hz, 1H, H-1.sup.B), 4.65 (d, J.sub.1,2=2.0 Hz, 1H, H-1.sup.A), 3.68 (s, 3H, —OCH.sub.3), 1.30 (d, J.sub.5,6=6.5 Hz, 3H, H-6.sup.A), 1.29 ppm (d, J.sub.5,6=6.2 Hz, 3H, H-6.sup.B); .sup.13C NMR (126 MHz, CDCl.sub.3): δ 174.0, 137.3, 137.1, 128.6, 128.5, 128.2, 128.1, 128.0, 98.8, 97.9, 78.5, 76.5, 72.9, 72.6, 72.1, 69.8, 67.5, 67.2, 67.1, 66.4, 64.6, 51.5, 33.9, 29.0, 25.7, 24.7, 18.5, 18.4 ppm; HRMS (ESI): m/z calcd for C.sub.33H.sub.44N.sub.6O.sub.9Na [M+Na].sup.+: 691.3062. found: 691.3054.

5′-Methoxycarbonylpentyl 4-azido-2,3-di-O-benzoyl-4,6-dideoxy-α-D-mannopyranosyl (1→3) 4-azido-2-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (71)

(133) A mixture of glycosyl donor 53 (0.124 g, 0.230 mmol), glycosyl acceptor 70 (0.140 g, 0.210 mmol) and freshly activated molecular sieves (3 Å, 0.5 g) in CH.sub.2Cl.sub.2 (4 mL) was stirred under argon for 5 h at room temperature. TMSOTf (8 μL, 0.046 mmol) was added and the resulting mixture was stirred for an additional hour. Then Et.sub.3N (1 mL) was added, the solid was filtered off and the residue was rinsed with CH.sub.2Cl.sub.2 (3×20 mL). The combined filtrate (80 mL) was washed with 20% aq. NaHCO.sub.3 (50 mL), water (30 mL), and brine (20 mL). The organic phase was separated, dried over MgSO.sub.4, and concentrated in vacuo. The residue was purified by column chromatography on silica gel (ethyl acetate-toluene gradient elution) to afford the title compound (0.210 g, 96%) as a white foam. Analytical data for 71: Rf=0.40 (ethyl acetate/toluene, 0.5/9.5, v/v); [M].sub.D.sup.21=−29.0 (c=1.2, CHCl.sub.3); .sup.1H NMR (500 MHz, CDCl.sub.3): δ 7.22-8.07 (m, 20H, H—Ar), 5.70 (dd, J.sub.2,3=3.4 Hz, 1H, H-2.sup.C), 5.59 (dd, J.sub.3,4=9.8 Hz, 1H, H-3.sup.C), 5.26 (d, J.sub.1,2=1.8 Hz, 1H, H-1.sup.C), 5.10 (d, J.sub.1,2=1.6 Hz, 1H, H-1.sup.B), 4.67 (d, J.sub.1,2=1.8 Hz, 1H, H-1.sup.A), 3.68 (s, 3H, —OCH.sub.3), 1.35 (d, J.sub.5,6=6.0 Hz, 3H, H-6.sup.C), 1.29 (d, J.sub.5,6=6.0 Hz, 3H, H-6.sup.A), 1.28 ppm (d, J.sub.5,6=6.0 Hz, 3H, H-6.sup.B); .sup.13C NMR (126 MHz, CDCl.sub.3): δ 174.0, 165.2, 165.0, 137.4, 137.3, 133.4, 133.2, 129.8 (×2), 129.4, 129.3, 128.6, 128.5 (×2), 128.3, 128.2, 128.1, 127.8, 127.6, 99.1, 98.8, 98.4, 78.4, 77.6, 76.1, 73.4, 72.4, 71.8, 70.7, 69.8, 68.2, 67.7, 67.5, 67.1, 64.7, 64.5, 63.3, 51.5, 33.9, 29.0, 25.7, 24.7, 18.6 (×2), 18.5 ppm; HRMS (ESI): m/z calcd for C.sub.53H.sub.61N.sub.9O.sub.14Na [M+Na].sup.+: 1070.4230. found: 1070.4249.

5′-Methoxycarbonylpentyl 4-azido-2,3-di-O-benzoyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→3) 4-azido-2-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (72)

(134) A mixture of glycosyl donor 63 (0.208 g, 0.296 mmol), glycosyl acceptor 70 (0.180 g, 0.269 mmol) and freshly activated molecular sieves (3 Å, 0.5 g) in CH.sub.2Cl.sub.2 (4 mL) was stirred under argon for 4 h at room temperature. MeOTf (213 μL, 1.88 mmol) was added and continued stirring for additional 48 h. Then Et.sub.3N (1 mL) was added, the solid was filtered off and the residue was rinsed with CH.sub.2Cl.sub.2 (3×20 mL). The combined filtrate (70 mL) was washed with 20% aq. NaHCO.sub.3 (40 mL), water (30 mL), and brine (20 mL). The organic phase was separated, dried over MgSO.sub.4, and concentrated in vacuo. The residue was purified by column chromatography on silica gel (ethyl acetate-toluene gradient elution) to afford the title compound (0.321 g, 91%) as a white foam. Analytical data for 72: Rf=0.30 (ethyl acetate/toluene, 0.5/9.5, v/v); [α].sub.D.sup.21=−17.9 (c=1.0, CHCl.sub.3); .sup.1H NMR (600 MHz, CDCl.sub.3): δ 7.05-8.00 (m, 25H, H—Ar), 5.71 (dd, J.sub.2,3=3.3 Hz, 1H, H-2.sup.D), 5.58 (dd, J.sub.3,4=10.3 Hz, 1H, H-3.sup.D), 5.09 (d, J.sub.1,2=1.5 Hz, 1H, H-1.sup.B), 5.07 (d, J.sub.1,2=1.6 Hz, 1H, H-1.sup.C), 5.04 (d, J.sub.1,2=1.6 Hz, 1H, H-1.sup.D), 1.31-1.35 (m, 6H, H-6.sup.A, H-6.sup.B), 1.26 ppm (d, J.sub.5,6=6.2 Hz, 3H, H-6.sup.C); .sup.13C NMR (126 MHz, CDCl.sub.3): δ 174.0, 165.2, 164.8, 137.5, 137.4, 137.3, 133.3, 133.2, 129.8, 129.7, 129.5, 129.3, 128.5 (×2), 128.4, 128.3, 128.2, 128.0, 127.8 (×2), 127.7, 127.6, 100.8, 99.2, 98.8, 98.3, 78.4, 77.9, 77.4, 76.4, 73.7, 73.2, 72.5, 72.1, 71.9, 70.9, 69.3, 68.1, 67.9, 67.7, 67.5, 67.2, 64.6, 63.6, 63.4, 51.5, 33.9, 29.1, 25.7, 24.7, 18.6 (×2), 18.5 ppm; HRMS (ESI): m/z calcd for C.sub.66H.sub.76N.sub.12O.sub.17Na [M+Na].sup.+: 1331.5344. found: 1331.5341.

5′-Methoxycarbonylpentyl 4-azido-4,6-dideoxy-α-D-mannopyranosyl (1→3) 4-azido-2-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (67a)

(135) Sodium methoxide (0.2 mL, 0.5 M solution) was added to a solution of 67 (0.350 g, 0.445 mmol) in CH.sub.3OH (5 mL) until pH ˜9 and the resulting mixture was stirred under argon for 4 h at room temperature. Then the reaction mixture was neutralized with Amberlite IR 120 (H.sup.+) ion exchange resin, the resin was filtered off and rinsed successively with CH.sub.3OH. The combined filtrate was concentrated in vacuo and purified by column chromatography on silica gel (ethyl acetate-toluene gradient elution) to afford the title compound (0.221 g, 86%) as a white foam. Analytical data for 67a: Rf=0.50 (CH.sub.3OH/CH.sub.2Cl.sub.2, 0.5/9.5, v/v); [α].sub.D.sup.21=+73.4 (c=1.0, CHCl.sub.3); .sup.1H NMR (500 MHz, CDCl.sub.3): δ 7.28-7.41 (m, 5H, H—Ar), 5.07 (s, 1H, H-1.sup.B), 4.78 (s, 1H, H-1.sup.A), 1.32 (d, J.sub.5,6=6.2 Hz, 3H, H-6.sup.A), 1.27 ppm (d, J.sub.5,6=6.2 Hz, 3H, H-6.sup.B); .sup.13C NMR (126 MHz, CDCl.sub.3): δ 174.1, 137.6, 128.5, 127.9, 127.6, 101.5, 96.9, 78.3, 76.9, 72.4, 70.3, 70.2, 67.7, 67.5, 67.3, 65.7, 64.7, 51.5, 33.9, 29.0, 25.7, 24.6, 18.5, 18.3 ppm; HRMS (ESI): m/z calcd for C.sub.26H.sub.38N.sub.6O.sub.9Na [M+Na].sup.+: 601.2592. found: 601.2581.

5′-Methoxycarbonylpentyl 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→3) 4,6-dideoxy-4-formamido-α-D-mannopyranoside (44)

(136) To a stirred solution of 67a (0.150 g, 0.259 mmol), in a pyridine (5 mL) and water (2 mL) mixture, H.sub.2S was bubbled for 0.5 h at 40° C., and then stirring was continued for 16 h. Then argon was bubbled through the solution for 10 min, solvents were removed in vacuo, and the residue was co-evaporated with toluene (3×10 mL) and dried. The high resolution mass spectrometry analysis showed completion of reaction to corresponding amine compound 67b and no products arising from incomplete reduction. HRMS (ESI): m/z calcd for C.sub.26H.sub.43N.sub.2O.sub.9 [M+H].sup.+: 527.2963. found: 527.2964. This crude material was directly used for formylation.

(137) Compound 67b in CH.sub.3OH (5 mL) at −20° C. was added a freshly prepared formic anhydride.sup.22 (5 mL, ethereal solution) and stirred for 3 h, then slowly allowed to warm to room temperature. Then solvents were evaporated and the residue was passed through column chromatography on silica gel (methanol-dichloromethane gradient elution) to afford disaccharide 67c. HRMS (ESI): m/z calcd for C.sub.28H.sub.42N.sub.2NaO.sub.11 [M+Na].sup.+: 605.2681. found: 605.2675.

(138) Compound 67c was dissolved in CH.sub.3OH/H.sub.2O (2:1, 6 mL), Pd(OH).sub.2 on carbon (20%, 0.050 g) was added. Then it was stirred under a pressure of hydrogen gas at room temperature for 16 h. After filtration through celite pad and washed with CH.sub.3OH (3×10 mL), and solvents were removed in vacuo. The residue was purified by column chromatography on silica gel (methanol-dichloromethane gradient elution) to afford the title compound (0.075 g, 59%, over 3 steps) as a white foam. Analytical data for 44: Rf=0.20 (CH.sub.3OH/CH.sub.2Cl.sub.2, 1.5/8.5, v/v); .sup.1H NMR (700 MHz, D.sub.2O): δ 8.21 ((d, J=15.4 Hz) and 8.03 (d, J=13.3 Hz), 2H, NCHO), 4.81-4.95 (m, 2H, 2×H-1), 3.70 (s, 3H, —OCH.sub.3), 1.20-1.30 ppm (m, 6H, 2×H-6); .sup.13C NMR (126 MHz, D.sub.2O): δ 178.5, 168.9, 168.8, 165.8, 165.7, 103.3, 103.2, 100.6, 100.5, 77.8 (×2), 70.2, 70.0, 69.9 (×2), 69.0, 68.9 (×2), 68.8 (×2), 68.6, 68.4, 68.2, 67.8, 57.6, 56.5, 53.1, 52.7, 52.6, 51.6, 34.6, 29.1, 25.9, 25.0, 17.8, 17.7, 17.6 ppm; HRMS (ESI): m/z calcd for C.sub.21H.sub.36N.sub.2O.sub.11Na [M+Na].sup.+: 515.2211. found: 515.2210.

(2′-Aminoethylamido)carbonylpentyl 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→3) 4,6-dideoxy-4-formamido-α-D-mannopyranoside (81)

(139) A solution of 44 (0.009 g, 0.018 mmol) in freshly distilled 1,2-diaminoethane (0.5 mL) was stirred at 65° C. for 48 h. Then excess reagent was removed in vacuo, and the residue was co-evaporated with CH.sub.3OH (3×10 mL) and dried. The residue was purified by reversed phase HPLC on C18 column in gradient water-acetonitrile and lyophilized, to give the title compound (0.0075 g, 79%) as a white foam. Analytical data for 81: Rf=0.15 (CH.sub.3OH/CH.sub.2Cl.sub.2, 1/1, v/v); .sup.1H NMR (700 MHz, D.sub.2O): δ 8.19-8.22 (Z) and 8.01-8.02 (E) (m, 2H, NCHO), 4.80-4.95 (m, 2H, 2×H-1), 1.19-1.30 ppm (m, 6H, 2×H-6); .sup.13C NMR (126 MHz, D.sub.2O): δ 178.4, 168.9, 168.8, 165.8, 165.7, 103.6, 103.3 (×2), 100.6, 100.5, 77.8, 77.4, 71.1, 70.4, 70.2, 70.0, 69.8, 69.0, 68.9, 68.8 (×3), 68.6, 68.4, 68.2, 67.8, 57.6, 56.5, 54.5, 52.6, 51.8, 51.6, 41.7, 41.4, 40.9, 40.7, 36.7, 29.2, 26.0, 25.9, 17.9, 17.8, 17.7 (×2) ppm; HRMS (ESI): m/z calcd for C.sub.22H.sub.40N.sub.4O.sub.10Na [M+Na].sup.+: 543.2637. found: 543.2642.

1-[(2′-Aminoethylamido)carbonylpentyl 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→3) 4,6-dideoxy-4-formamido-α-D-mannopyranoside]-2-butoxycyclobutene-3,4-dione (87)

(140) To a stirred solution of 81 (0.0075 g, 0.014 mmol) in water (0.5 mL) and EtOH (0.4 mL), a solution of 3,4-dibutoxy-3-cyclobutene-1,2-dione (20% in ethanol, 70 μL) was added and the pH was adjusted to 8 by careful addition of aq.NaHCO.sub.3 (1%) solution. After 0.5 h, TLC showed the reaction was complete; the reaction mixture was neutralized using CH.sub.3COOH (10%) and concentrated in vacuo. The residue was purified by reversed phase HPLC on a C18 column with a gradient of water-acetonitrile and lyophilized, to give the title compound (0.0089 g, 92%) as a white foam. Analytical data for 87: Rf=0.20 (CH.sub.3OH/CH.sub.2Cl.sub.2, 1.5/8.5, v/v); .sup.1H NMR (500 MHz, D.sub.2O): δ 8.26-8.30 (Z) and 8.09-8.12 (E) (m, 2H, NCHO), 4.83-5.03 (m, 2H, 2×H-1), 1.26-1.37 (m, 6H, 2×H-6), 0.99-1.05 ppm (m, 3H, —CH.sub.31); .sup.13C NMR (126 MHz, D.sub.2O): δ 189.8, 189.6, 184.3 (×2), 178.5, 178.1, 178.0, 177.9, 174.8, 174.7, 168.9, 168.8, 165.8, 165.7, 103.3, 103.2, 100.5 (×2), 77.8, 75.4, 75.3, 70.2, 70.0, 69.9, 69.0, 68.9 (×2), 68.8, 68.7, 68.6, 68.4, 68.2, 67.7, 57.6, 56.5, 52.7, 52.6, 51.7, 45.2, 45.0, 40.3, 40.2, 36.7, 32.4, 31.2, 29.3 (×2), 26.2, 26.1, 25.9 (×2), 19.1, 19.0, 17.8 (×2), 17.7, 13.9 ppm; HRMS (ESI): m/z calcd for C.sub.30H.sub.48N.sub.4O.sub.13Na [M+Na].sup.+: 695.3110. found: 695.3113.

5′-Methoxycarbonylpentyl 4-azido-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→3) 4-azido-2-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (68a)

(141) Sodium methoxide (0.2 mL, 0.5 M solution) was added to a solution of 68 (0.352 g, 0.336 mmol) in CH.sub.3OH (5 mL) until pH ˜9 and the resulting mixture was stirred under argon for 4 h at room temperature. Then the reaction mixture was neutralized with Amberlite IR 120 (H.sup.+) ion exchange resin, the resin was filtered off and rinsed successively with CH.sub.3OH. The combined filtrate was concentrated in vacuo and purified by column chromatography on silica gel (ethyl acetate-toluene gradient elution) to afford the title compound (0.256 g, 91%) as an off-white foam. Analytical data for 68a: Rf=0.30 (ethyl acetate/toluene, 1/4, v/v); [α].sub.D.sup.21=+72.4 (c=1.0, CHCl.sub.3); .sup.1H NMR (500 MHz, CDCl.sub.3): δ 7.27-7.39 (m, 10H, H—Ar), 5.02 (s, 1H, H-1.sup.B), 4.93 (s, 1H, H-1.sup.C), 4.77 (s, 1H, H-1.sup.A), 3.67 (s, 3H, —OCH.sub.3), 1.24 ppm (d, J.sub.5,6=6.2 Hz, 3H, H-6.sup.C); .sup.13C NMR (126 MHz, CDCl.sub.3): δ 174.0, 137.6, 137.4, 128.6, 128.5, 128.1, 127.9, 127.5, 101.0 (×2), 97.0, 78.3, 77.8, 73.1, 72.5, 72.1, 70.2, 69.9, 67.9, 67.6, 67.4, 67.3, 65.8, 64.8, 64.0, 51.5, 33.9, 29.0, 25.6, 24.6, 18.6, 18.5, 18.3 ppm; HRMS (ESI): m/z calcd for C.sub.39H.sub.53N.sub.9O.sub.12Na [M+Na].sup.+: 862.3706. found: 862.3691.

5′-Methoxycarbonylpentyl 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→3) 4,6-dideoxy-4-formamido-α-D-mannopyranoside (44)

(142) To a stirred solution of 68a (0.114 g, 0.136 mmol), in pyridine (5 mL) and water (2 mL) mixture, H.sub.2S was bubbled for 0.5 h at 40° C., and then stirring was continued for 16 h. Then argon was bubbled through the solution for 10 min, solvents were removed in vacuo, and the residue was co-evaporated with toluene (3×10 mL) and dried. The high resolution mass spectrometry analysis showed completion of reaction to corresponding amine compound 68b and no products arising from incomplete reduction. HRMS (ESI): m/z calcd for C.sub.39H.sub.60N.sub.3O.sub.12 [M+H].sup.+: 762.4172. found: 762.4171. This crude material was directly used for formylation.

(143) Compound 68b in CH.sub.3OH (5 mL) at −20° C. was added a freshly prepared formic anhydrid.sup.22 (5 mL, ethereal solution) and stirred for 3 h, then slowly allowed to warm to room temperature. Then solvents were evaporated and the residue was passed through column chromatography on silica gel (methanol-dichloromethane gradient elution) to afford disaccharide 68c. HRMS (ESI): m/z calcd for C.sub.42H.sub.59N.sub.3O.sub.15Na [M+Na].sup.+: 868.3838. found: 868.3827.

(144) Compound 68c was dissolved in CH.sub.3OH/H.sub.2O (2:1, 5 mL), Pd(OH).sub.2 on carbon (20%, 0.040 g) was added. Then it was stirred under a pressure of hydrogen gas at room temperature for 16 h. After filtration through celite pad and washed with CH.sub.3OH (3×10 mL), and solvents were removed in vacuo. The residue was purified by column chromatography on silica gel (methanol-dichloromethane gradient elution) to afford the title compound (0.046 g, 51%, over 3 steps) as a white foam. Analytical data for 45: Rf=0.40 (CH.sub.3OH/CH.sub.2Cl.sub.2, 3/7, v/v); .sup.1H NMR (700 MHz, D.sub.2O): a 8.20-8.24 (Z) and 8.02-8.06 (E) (m, 3H, NCHO), 4.82-5.08 (m, 3H, 3×H-1), 1.21-1.32 ppm (m, 9H, 3×H-6); .sup.13C NMR (126 MHz, D.sub.2O): δ 178.5, 168.8, 168.7, 165.8 (×2), 165.6 (×2), 103.4, 103.3, 102.9, 101.8, 101.7, 100.6, 100.5, 79.0 (×2), 78.9, 78.2, 78.0, 77.7 (×3), 70.0, 69.9, 69.8, 69.4, 69.2, 69.0, 68.9 (×2), 68.8 (×2), 68.7, 68.6 (×2), 68.5 (×2), 68.3 (×2), 67.9, 67.7, 57.8, 57.7, 56.4, 53.1, 52.8, 52.7, 51.9, 34.6, 29.1, 25.9, 25.0, 18.1, 17.9 (×3), 17.8, 17.7 ppm; HRMS (ESI): m/z calcd for C.sub.28H.sub.47N.sub.3O.sub.15Na [M+Na].sup.+: 688.2899. found: 688.2895.

(2′-Aminoethylamido)carbonylpentyl 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→3) 4,6-dideoxy-4-formamido-α-D-mannopyranoside (82)

(145) A solution of 45 (0.012 g, 0.018 mmol) in freshly distilled 1,2-diaminoethane (0.5 mL) was stirred at 65° C. for 48 h. Then excess reagent was removed in vacuo, and the residue was co-evaporated with CH.sub.3OH (3×10 mL) and dried. The residue was purified by reversed phase HPLC on a C18 column with a gradient of water-acetonitrile and lyophilized, to give the title compound (0.0112 g, 90%) as a white foam. Analytical data for 82: Rf=0.10 (CH.sub.3OH/CH.sub.2Cl.sub.2, 1/1, v/v); .sup.1H NMR (500 MHz, D.sub.2O): δ 8.26-8.31 (Z) and 8.08-8.13 (E) (m, 3H, NCHO), 4.88-5.16 (m, 3H, 3×H-1), 1.20-1.37 (m, 9H, 3×H-6); .sup.13C NMR (126 MHz, D.sub.2O): δ 178.4, 178.2, 168.8 (×2), 165.9, 165.8, 165.6 (×3), 103.3 (×2), 101.7 (×2), 100.5 (×2), 79.0, 77.7, 70.0, 69.9, 69.2, 69.0, 68.9, 68.8, 68.6, 68.4, 68.0, 67.7, 57.8, 57.7, 52.8, 52.7, 51.9, 41.5 (×2), 41.5, 41.4, 40.9, 40.6 (×2), 36.6, 29.2, 26.0 (×2), 25.9, 25.8, 18.1, 18.0, 17.9 (×3), 17.7 ppm; HRMS (ESI): m/z calcd for C.sub.29H.sub.51N.sub.5O.sub.14Na [M+Na].sup.+: 716.3325. found: 716.3311.

1-[(2′-Aminoethylamido)carbonylpentyl 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→3) 4,6-dideoxy-4-formamido-α-D-mannopyranoside]-2-butoxycyclobutene-3,4-dione (88)

(146) To a stirred solution of 82 (0.0075 g, 0.011 mmol) in water (0.5 mL) and EtOH (0.4 mL), a solution of 3,4-dibutoxy-3-cyclobutene-1,2-dione (20% in ethanol, 50 μL) was added and the pH was adjusted to 8 by careful addition of aq.NaHCO.sub.3 (1%) solution. After 0.5 h, TLC showed the reaction was complete; the reaction mixture was neutralized using CH.sub.3COOH (10%) and concentrated in vacuo. The residue was purified by reversed phase HPLC on a C18 column with a gradient of water-acetonitrile and lyophilized, to give the title compound (0.0065 g, 71%) as a white foam. Analytical data for 88: Rf=0.20 (CH.sub.3OH/CH.sub.2Cl.sub.2, 1/4, v/v); .sup.1H NMR (500 MHz, D.sub.2O): δ 8.27-8.31 (Z) and 8.08-8.13 (E) (m, 3H, NCHO), 4.86-5.15 (m, 3H, 3×H-1), 1.26-1.36 (m, 9H, 3×H-6), 0.98-1.05 ppm (m, 3H, —CH.sub.31); .sup.13C NMR (126 MHz, D.sub.2O): δ 189.8, 189.6, 184.3 (×2), 178.5, 178.1, 178.0, 177.9, 174.8, 174.7, 168.8, 168.7, 165.8 (×2), 165.6, 103.4, 103.3, 102.9, 101.8, 101.7, 100.6, 100.5, 79.0, 78.9, 78.8, 78.2, 78.0, 77.7 (×2), 75.4, 75.3, 70.0, 69.9, 69.3, 69.2, 69.0, 68.9, 68.9, 68.8, 68.6, 68.5 (×2), 68.4, 68.3, 67.9, 67.7, 57.8, 57.7, 56.5, 52.8, 52.8, 52.7, 51.9, 45.2, 45.0, 40.3, 40.2, 36.7, 32.4, 29.3 (×2), 26.2, 26.1, 25.9 (×2), 19.1, 19.0, 18.1, 18.0, 17.9 (×3), 17.7, 13.9 ppm; HRMS (ESI): m/z calcd for C.sub.37H.sub.59N.sub.5O.sub.17Na [M+Na].sup.+: 868.3798. found: 868.3800.

5′-Methoxycarbonylpentyl 4-azido-4,6-dideoxy-α-D-mannopyranosyl (1→3) 4-azido-2-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (71a)

(147) Sodium methoxide (0.5 mL, 0.5 M solution) was added to a solution of 71 (0.458 g, 0.437 mmol) in CH.sub.3OH (10 mL) until pH −9 and the resulting mixture was stirred under argon for 4 h at room temperature. Then the reaction mixture was neutralized with Amberlite IR 120 (H.sup.+) ion exchange resin, the resin was filtered off and rinsed successively with CH.sub.3OH. The combined filtrate was concentrated in vacuo and purified by column chromatography on silica gel (ethyl acetate-toluene gradient elution) to afford the title compound (0.341 g, 93%) as oil. Analytical data for 71a: Rf=0.30 (ethyl acetate/toluene, 1/4, v/v); [α].sub.D.sup.21=+58.9 (c=1.3, CHCl.sub.3); .sup.1H NMR (500 MHz, CDCl.sub.3): δ 7.12-7.39 (m, 10H, H—Ar), 5.08 (d, J.sub.1,2=1.8 Hz, 1H, H-1.sup.B), 5.05 (d, J.sub.1,2=1.6 Hz, 1H, H-1.sup.C), 4.66 (d, J.sub.1,2=1.6 Hz, 1H, H-1.sup.A), 3.68 (s, 3H, —OCH.sub.3), 1.28-1.32 (m, 6H, H-6.sup.A, H-6.sup.B), 1.19 ppm (d, J.sub.5,6=6.2 Hz, 3H, H-6.sup.C); .sup.13C NMR (126 MHz, CDCl.sub.3): δ 174.1, 137.3 (×2), 128.5, 128.4, 128.2, 128.1, 127.8, 127.6, 101.5, 98.8, 98.1, 78.4, 77.5, 76.4, 73.2, 72.6, 71.7, 70.1, 70.0, 67.9, 67.5, 67.4, 67.2, 65.5, 64.7, 64.4, 51.5, 33.9, 29.0, 25.7, 24.6, 18.6, 18.5, 18.2 ppm; HRMS (ESI): m/z calcd for C.sub.39H.sub.53N.sub.9O.sub.12Na [M+Na].sup.+: 862.3706. found: 862.3700.

5′-Methoxycarbonylpentyl 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→3) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranoside (46)

(148) To a stirred solution of 71a (0.157 g, 0.187 mmol), in pyridine (5 mL) and water (2 mL) mixture, H.sub.2S was bubbled for 0.5 h at 40° C., and then stirring was continued for 16 h. Then argon was bubbled through the solution for 10 min, solvents were removed in vacuo, and the residue was co-evaporated with toluene (3×10 mL) and dried. The high resolution mass spectrometry analysis showed completion of reaction to corresponding amine compound 71b and no products arising from incomplete reduction. HRMS (ESI): m/z calcd for C.sub.39H.sub.60N.sub.3O.sub.12 [M+H].sup.+: 762.4172. found: 762.4182. This crude material was directly used for formylation.

(149) Compound 71b in CH.sub.3OH (5 mL) at −20° C. was added a freshly prepared formic anhydride.sup.22 (5 mL, ethereal solution) and stirred for 3 h, then slowly allowed to warm to room temperature. Then solvents were evaporated and the residue was passed through column chromatography on silica gel (methanol-dichloromethane gradient elution) to afford disaccharide 71c. HRMS (ESI): m/z calcd for C.sub.42H.sub.59N.sub.3O.sub.15Na [M+Na].sup.+: 868.3838. found: 868.3834.

(150) Compound 71c was dissolved in CH.sub.3OH/H.sub.2O (2:1, 5 mL), Pd(OH).sub.2 on carbon (20%, 0.050 g) was added. Then it was stirred under a pressure of hydrogen gas at room temperature for 16 h. After filtration through celite pad and washed with CH.sub.3OH (3×10 mL), and solvents were removed in vacuo. The residue was purified by column chromatography on silica gel (methanol-dichloromethane gradient elution) to afford the title compound (0.053 g, 43%, over 3 steps) as a white foam. Analytical data for 46: Rf=0.50 (CH.sub.3OH/CH.sub.2Cl.sub.2, 3/7, v/v); .sup.1H NMR (700 MHz, D.sub.2O): δ 8.20-8.24 (Z) and 8.02-8.06 (E) (m, 3H, NCHO), 4.92-5.04 (m, 3H, 3×H-1), 3.70 (s, 3H, —OCH.sub.3), 1.22-1.31 ppm (m, 9H, 3×H-6); .sup.13C NMR (176 MHz, D.sub.2O): δ 178.3, 168.7, 168.6, 168.5, 165.6 (×2), 165.5, 103.1, 102.9 (×2), 102.8, 102.7 (×2), 99.0 (×3), 78.6 (×2), 78.5, 77.3, 77.2, 77.1, 70.1, 70.0, 69.8, 69.5, 69.4, 68.9, 68.8 (×2), 68.7 (×2), 68.6 (×2), 68.5 (×2), 68.4 (×2), 68.3, 68.2, 67.8, 57.7, 57.4, 56.3, 52.9, 52.8, 52.4, 51.3, 34.4, 28.9, 25.7, 24.8, 17.6 (×2), 17.5, 17.4 ppm; HRMS (ESI): m/z calcd for C.sub.28H.sub.47N.sub.3O.sub.15Na [M+Na].sup.+: 688.2899. found: 688.2893.

(2′-Aminoethylamido)carbonylpentyl 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→3) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranoside (83)

(151) A solution of 46 (0.010 g, 0.015 mmol) in freshly distilled 1,2-diaminoethane (0.5 mL) was stirred at 65° C. for 48 h. Then excess reagent was removed in vacuo, and the residue was co-evaporated with CH.sub.3OH (3×10 mL) and dried. The residue was purified by reversed phase HPLC on a C18 column with a gradient of water-acetonitrile and lyophilized, to give the title compound (0.0084 g, 81%) as a white foam. Analytical data for 83: Rf=0.10 (CH.sub.3OH/CH.sub.2Cl.sub.2, 1/1, v/v); .sup.1H NMR (500 MHz, D.sub.2O): δ 8.26-8.31 (Z) and 8.09-8.13 (E) (m, 3H, NCHO), 4.98-5.11 (m, 3H, 3×H-1), 1.26-1.38 ppm (m, 9H, 3×H-6); .sup.13C NMR (126 MHz, D.sub.2O): δ 177.7, 177.2, 168.0, 167.9, 167.8, 164.9, 164.8, 164.6, 102.4, 102.2 (×2), 102.1, 101.9 (×2), 98.3, 98.2, 77.9, 77.8 (×3), 76.8, 76.5 (×2), 76.4, 76.2, 70.0, 69.9, 69.5, 69.3, 69.0, 68.8 (×2), 68.7, 68.2, 68.1, 68.0 (×2), 67.9, 67.8 (×2), 67.7, 67.6 (×2), 67.5, 67.1, 57.0, 56.7, 55.5, 53.5, 52.0, 51.7 (×2), 51.6, 50.7, 50.6 (×2), 40.4, 39.9, 39.5, 39.0, 35.7, 35.6, 28.2, 25.1, 24.9 (×2), 17.0, 16.9, 16.7 (×2) ppm; HRMS (ESI): m/z calcd for C.sub.29H.sub.51N.sub.5O.sub.14Na [M+Na].sup.+: 716.3325. found: 716.3322.

1-[(2′-Aminoethylamido)carbonylpentyl 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→3) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranoside]-2-butoxycyclobutene-3,4-dione (89)

(152) To a stirred solution of 83 (0.0074 g, 0.0106 mmol) in water (0.5 mL) and EtOH (0.4 mL), a solution of 3,4-dibutoxy-3-cyclobutene-1,2-dione (20% in ethanol, 50 μL) was added and pH was adjusted to 8 by careful addition of aq.NaHCO.sub.3 (1%) solution. After 0.5 h, TLC showed the reaction was complete; the reaction mixture was neutralized using CH.sub.3COOH (10%) and concentrated in vacuo. The residue was purified by reversed phase HPLC on a C18 column with a gradient of water-acetonitrile and lyophilized, to give the title compound (0.0072 g, 80%) as a white foam. Analytical data for 89: Rf=0.20 (CH.sub.3OH/CH.sub.2Cl.sub.2, 1/4, v/v); .sup.1H NMR (500 MHz, D.sub.2O): δ 8.16-8.21 (Z) and 7.99-8.03 (E) (m, 3H, NCHO), 4.87-5.01 (m, 3H, 3×H-1), 1.18-1.27 (m, 9H, 3×H-6); .sup.13C NMR (126 MHz, D.sub.2O): δ 189.8, 189.6, 184.2, 178.5, 178.0, 177.9 (×2), 174.8, 174.7, 168.8, 168.7, 166.6, 165.8, 165.7, 103.1, 102.8, 99.1 (×2), 78.7, 77.4 (×2), 75.4, 75.3, 70.2, 69.7, 69.6, 69.1, 69.0, 68.9, 68.7 (×3), 68.5, 68.4, 53.0, 52.6, 51.5, 45.1, 44.9, 40.3, 40.1, 36.7, 32.4, 29.2, 26.1, 26.0, 25.9 (×2), 19.0 (×2), 17.8 (×3), 17.6, 13.9 ppm; HRMS (ESI): m/z calcd for C.sub.37H.sub.59N.sub.5O.sub.17Na [M+Na].sup.+: 868.3798. found: 868.3791.

5′-Methoxycarbonylpentyl 4-azido-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→3) 4-azido-2-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (72a)

(153) Sodium methoxide (0.5 mL, 0.5 M solution) was added to a solution of 72 (0.391 g, 0.299 mmol) in CH.sub.3OH/THF mixture (4:1, 15 mL) until pH ˜9 and the resulting mixture was stirred under argon for 5 h at room temperature. Then the reaction mixture was neutralized with Amberlite IR 120 (H.sup.+) ion exchange resin, the resin was filtered off and rinsed successively with CH.sub.3OH. The combined filtrate was concentrated in vacuo and purified by column chromatography on silica gel (ethyl acetate-toluene gradient elution) to afford the title compound (0.311 g, 95%) as oil. Analytical data for 72a: Rf=0.40 (ethyl acetate/toluene, 1/4, v/v); [α].sub.D.sup.21=+51.2 (c=1.0, CHCl.sub.3); .sup.1H NMR (500 MHz, CDCl.sub.3): δ 7.10-7.39 (m, 15H, H—Ar), 5.07 (d, J.sub.1,2=1.6 Hz, 1H, H-1.sup.B), 5.02 (d, J.sub.1,2=1.6 Hz, 1H, H-1.sup.C), 4.91 (d, J.sub.1,2=1.5 Hz, 1H, H-1.sup.D), 4.66 (d, J.sub.1,2=2.0 Hz, 1H, H-1.sup.A), 3.68 (s, 3H, —OCH.sub.3), 1.26-1.35 (m, 9H, H-6.sup.A, H-6.sup.B H-6.sup.C), 1.17 ppm (d, J.sub.5,6=5.9 Hz, 3H, H-6.sup.D); .sup.13C NMR (126 MHz, CDCl.sub.3): δ 174.0, 137.5, 137.4, 137.3, 128.5, 128.4, 128.2, 128.0 (×2), 127.8, 127.5, 101.0, 100.8, 98.8, 98.2, 78.4, 77.9, 77.5, 76.4, 73.2, 72.5, 72.1, 71.8, 70.0, 69.7, 67.9 (×2), 67.5 (×2), 67.1, 65.8 (×2), 64.7, 64.6, 63.9, 51.5, 33.9, 29.0, 25.7, 24.7, 18.6 (×2), 18.5, 18.3 ppm; HRMS (ESI): m/z calcd for C.sub.52H.sub.68N.sub.12O.sub.15Na [M+Na].sup.+: 1123.4806. found: 1123.4812.

5′-Methoxycarbonylpentyl 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→3) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranoside (47)

(154) To a stirred solution of 72a (0.146 g, 0.132 mmol), in pyridine (5 mL) and water (2 mL) mixture, H.sub.2S was bubbled for 0.5 h at 40° C., and then stirring was continued for 16 h. Then argon was bubbled through the solution for 10 min, solvents were removed in vacuo, and the residue was co-evaporated with toluene (3×10 mL) and dried. The high resolution mass spectrometry analysis showed completion of reaction to corresponding amine compound 72b and no products arising from incomplete reduction. HRMS (ESI): m/z calcd for C.sub.52H.sub.77N.sub.4O.sub.15 [M+H].sup.+: 997.5380. found: 997.5366. This crude material was directly used for formylation.

(155) Compound 72b in CH.sub.3OH (5 mL) at −20° C. was added a freshly prepared formic anhydride Olah et al. (Angew (1979) Chem. Int. Ed. 18, 614) (5 mL, ethereal solution) and stirred for 3 h, then slowly allowed to warm to room temperature. Then solvents were evaporated and the residue was passed through column chromatography on silica gel (methanol-dichloromethane gradient elution) to afford disaccharide 72c. HRMS (ESI): m/z calcd for C.sub.56H.sub.76N.sub.4O.sub.19Na [M+Na].sup.+: 1131.4996. found: 1131.4992.

(156) Compound 72c was dissolved in CH.sub.3OH/H.sub.2O (2:1, 5 mL), Pd(OH).sub.2 on carbon (20%, 0.050 g) was added. Then it was stirred under a pressure of hydrogen gas at room temperature for 16 h. After filtration through celite pad and washed with CH.sub.3OH (3×10 mL), and solvents were removed in vacuo. The residue was purified by column chromatography on silica gel (methanol dichloromethane gradient elution) to afford the title compound (0.068 g, 61%, over 3 steps) as a white foam. Analytical data for 47: Rf=0.30 (CH.sub.3OH/CH.sub.2Cl.sub.2, 3/7, v/v); .sup.1H NMR (500 MHz, D.sub.2O): δ 8.26-8.33 (Z) and 8.06-8.14 (E) (m, 4H, NCHO), 4.98-5.20 (m, 4H, 4×H-1), 3.85-4.28 (m, 16H, 4×H-2, 4×H-3, 4×H-4, 4×H-5), 1.28-1.39 ppm (m, 12H, 4×H-6); .sup.13C NMR (126 MHz, D.sub.2O): δ 178.5, 168.8, 168.7, 168.6, 165.8, 165.6, 103.3, 103.2, 102.9, 102.8, 102.7, 101.9, 101.6, 99.3, 99.2, 79.3, 78.9, 78.7, 78.5, 78.4, 78.1, 77.3, 69.9 (×2), 69.8, 69.7, 69.4, 69.3, 69.0, 68.9, 68.8 (×2), 68.7, 68.6, 68.5, 68.4, 68.3, 68.0, 58.0, 57.7, 56.5, 53.1, 52.9, 52.8, 52.7, 51.8, 34.6, 29.1, 25.9, 25.0, 18.1 (×2), 18.0 (×2), 17.9 (×2), 17.8 (×2), 17.7 (×2) ppm; HRMS (ESI): m/z calcd for C.sub.35H.sub.58N.sub.4O.sub.19Na [M+Na].sup.+: 861.3587. found: 861.3580.

(2′-Aminoethylamido)carbonylpentyl 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→3) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranoside (84)

(157) A solution of 47 (0.0134 g, 0.016 mmol) in freshly distilled 1,2-diaminoethane (0.5 mL) was stirred at 65° C. for 48 h. Then excess reagent was removed in vacuo, and the residue was co-evaporated with CH.sub.3OH (3×10 mL) and dried. The residue was purified by reversed phase HPLC on a C18 column with a gradient of water-acetonitrile and lyophilized, to give the title compound (0.0113 g, 82%) as a white foam. Analytical data for 84: Rf=0.10 (CH.sub.3OH/CH.sub.2Cl.sub.2, 1/1, v/v); .sup.1H NMR (500 MHz, D.sub.2O): δ 8.16-8.23 (Z) and 7.98-8.05 (E) (m, 4H, NCHO), 4.84-5.10 (m, 4H, 4×H-1), 2.22-2.28 (m, 2H, —CH.sub.2f—), 1.54-1.64 (m, 4H, —CH.sub.2e—, —CH.sub.2c—), 1.30-1.41 (m, 2H, —CH.sub.2d—), 1.18-1.30 ppm (m, 12H, 4×H-6); .sup.13C NMR (125 MHz, D.sub.2O): δ 177.4, 167.9 (×2), 164.9, 164.6, 102.6, 102.3 (×3), 101.9 (×2), 101.0 (×2), 100.8, 98.6, 98.3, 78.1, 77.6, 77.5 (×2), 76.8, 76.4, 76.0, 70.3, 70.2, 70.1, 70.0 (×2), 69.5, 69.2, 68.9 (×3), 68.3, 68.0, 67.8, 67.6 (×2), 53.9, 53.8, 53.6, 52.0, 51.8, 50.8 (×2), 40.6 (×3), 39.7, 35.7, 28.2, 25.0, 24.9 (×2), 17.3, 17.0, 16.8 (×3) ppm; HRMS (ESI): m/z calcd for C.sub.36H.sub.62N.sub.6O.sub.18Na [M+Na].sup.+: 889.4013. found: 889.4020.

1-[(2′-Aminoethylamido)carbonylpentyl 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→3) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranoside]-2-butoxycyclobutene-3,4-dione (90)

(158) To a stirred solution of 84 (0.013 g, 0.015 mmol) in water (0.5 mL) and EtOH (0.4 mL), a solution of 3,4-dibutoxy-3-cyclobutene-1,2-dione (20% in ethanol, 70 μL) was added and pH was adjusted to 8 by careful addition of aq.NaHCO.sub.3 (1%) solution. After 0.5 h, TLC showed the reaction was complete; the reaction mixture was neutralized using CH.sub.3COOH (10%) and concentrated in vacuo. The residue was purified by reversed phase HPLC on a C18 column with a gradient of water-acetonitrile and lyophilized, to give the title compound (0.0109 g, 72%) as a white foam. Analytical data for 90: Rf=0.20 (CH.sub.3OH/CH.sub.2C2, 1/4, v/v); .sup.1H NMR (600 MHz, D.sub.2O): δ 8.18-8.24 (Z) and 7.98-8.06 (E) (m, 4H, NCHO), 4.90-5.10 (m, 4H, 4×H-1), 1.20-1.35 (m, 14H, —CH.sub.2d—, 4×H-6) ppm; .sup.13C NMR (126 MHz, D.sub.2O): δ 189.8, 189.6, 184.3 (×2), 178.5, 178.2, 178.1, 178.0, 177.9, 174.8, 174.7, 168.8, 168.7, 168.6, 165.8, 165.6, 103.3 (×2), 103.2, 102.8 (×2), 102.7, 101.7 (×2), 101.6 (×2), 99.2, 78.9 (×2), 78.4, 78.1, 77.3, 75.4, 75.3, 69.9 (×2), 69.8 (×2), 69.7, 69.3, 69.0 (×2), 68.9, 68.8 (×4), 68.6 (×2), 68.5, 68.4 (×2), 68.0, 62.5, 52.9, 52.8 (×2), 52.7, 51.8, 45.2, 45.0, 44.2, 40.6, 40.3, 40.2, 36.7, 34.4, 32.4, 29.2, 26.2, 26.1, 26.0 (×2), 25.9 (×2), 25.8, 19.3, 19.1, 19.0, 18.1, 18.0 (×2), 17.9, 17.8 (×2), 17.7 (×2), 14.0, 13.9 ppm; HRMS (ESI): m/z calcd for C.sub.44H.sub.70N.sub.6O.sub.21Na [M+Na].sup.+: 1041.4486. found: 1041.4484.

5′-Methoxycarbonylpentyl 4-azido-2-O-benzoyl-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (73)

(159) A mixture of glycosyl donor 64 (0.743 g, 1.08 mmol), glycosyl acceptor 66 Cheng. et al. (2010) Angew. Chem. Int. Ed. 49, 4771-4774) (0.400 g, 0.982 mmol) and freshly activated molecular sieves (3 Å, 1.5 g) in CH.sub.2Cl.sub.2 (10 mL) was stirred under argon for 4 h at room temperature. MeOTf (0.890 mL, 7.86 mmol) was added and stirring was continued for an additional 48 h. Then Et.sub.3N (1 mL) was added, the solid was filtered off and the residue was rinsed with CH.sub.2Cl.sub.2 (3×30 mL). The combined filtrate (100 mL) was washed with 20% aq. NaHCO.sub.3 (40 mL), water (30 mL), and brine (20 mL). The organic phase was separated, dried over MgSO.sub.4, and concentrated in vacuo. The residue was purified by column chromatography on silica gel (ethyl acetate-toluene gradient elution) to afford the title compound (0.865 g, 85%) as oil. Analytical data for 73: Rf=0.50 (ethyl acetate/toluene, 0.5/9.5, v/v); [α].sub.D.sup.21=+36.7 (c=1.3, CHCl.sub.3); .sup.1H NMR (500 MHz, CDCl.sub.3): δ 7.15-8.11 (m, 20H, H—Ar), 5.61 (dd, J.sub.2,3=3.0 Hz, 1H, H-2.sup.C), 4.99 (d, J.sub.1,2=1.8 Hz, 1H, H-1.sup.B), 4.90 (d, J.sub.1,2=2.0 Hz, 1H, H-1.sup.C), 4.64 (s, 1H, H-1.sup.A), 3.70 (s, 3H, —OCH.sub.3), 1.30 (d, J.sub.5,6=6.0 Hz, 6H, H-6.sup.B, H-6.sup.C), 1.25 ppm (d, J.sub.5,6=6.0 Hz, 3H, H-6.sup.A); .sup.13C NMR (126 MHz, CDCl.sub.3): δ 174.0, 165.3, 137.4, 137.3, 137.2, 133.3, 129.9, 129.8, 128.5 (×3), 128.4, 128.3, 128.1, 128.0 (×2), 127.9, 100.4, 99.2, 98.7, 77.6, 76.7, 75.4, 74.2, 74.1, 72.2, 72.1, 71.4, 67.7 (×2), 67.5, 67.1, 64.4, 64.1 (×2), 51.5, 33.9, 29.1, 25.7, 24.7, 18.7, 18.6 ppm; HRMS (ESI): m/z calcd for C.sub.53H.sub.63N.sub.9O.sub.13Na [M+Na].sup.+: 1056.4438. found: 1056.4436.

5′-Methoxycarbonylpentyl 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (74)

(160) Sodium methoxide (0.7 mL, 0.5 M solution) was added to a solution of 73 (0.855 g, 0.827 mmol) in CH.sub.3OH (10 mL) until pH ˜9 and the resulting mixture was stirred under argon for 4 h at room temperature. Then the reaction mixture was neutralized with Amberlite IR 120 (H.sup.+) ion exchange resin, the resin was filtered off and rinsed successively with CH.sub.3OH. The combined filtrate was concentrated in vacuo and purified by column chromatography on silica gel (ethyl acetate-toluene gradient elution) to afford the title compound (0.720 g, 94%) as oil. Analytical data for 74: Rf=0.30 (ethyl acetate/toluene, 0.5/9.5, v/v); [α].sub.D.sup.21=+94.4 (c=1.1, CHCl.sub.3); .sup.1H NMR (500 MHz, CDCl.sub.3): δ 7.29-7.45 (m, 15H, H—Ar), 4.95 (br. s., 2H, H-1.sup.B, H-1.sup.C), 4.63 (d, J.sub.1,2=2.0 Hz, 1H, H-1.sup.A), 3.70 (s, 3H, —OCH.sub.3), 1.28-1.31 (m, 6H, H-6.sup.B, H-6.sup.C), 1.21 ppm (d, J.sub.5,6=6.2 Hz, 3H, H-6.sup.A); .sup.13C NMR (126 MHz, CDCl.sub.3): δ 174.0, 137.4, 137.3, 137.1, 128.6 (×2), 128.3 (×2), 128.2 (×3), 128.1, 100.5 (×2), 98.7, 77.6, 77.5, 76.9, 74.0, 73.3, 72.2, 72.1 (×2), 67.7, 67.5, 67.3, 67.2, 67.1, 64.4, 64.2, 63.8, 51.5, 33.9, 29.0, 25.7, 24.7, 18.6 (×2), 18.3 ppm; HRMS (ESI): m/z calcd for C.sub.46H.sub.59N.sub.9O.sub.12Na [M+Na].sup.+: 952.4175. found: 952.4176.

5′-Methoxycarbonylpentyl 4-azido-2-O-benzoyl-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (75)

(161) A mixture of glycosyl donor 64 (0.262 g, 0.381 mmol), glycosyl acceptor 74 (0.322 g, 0.346 mmol) and freshly activated molecular sieves (3 Å, 0.5 g) in CH.sub.2C.sub.2 (8 mL) was stirred under argon for 4 h at room temperature. MeOTf (320 μL, 2.77 mmol) was added and stirring was continued for an additional 48 h. Then Et.sub.3N (1 mL) was added, the solid was filtered off and the residue was rinsed with CH.sub.2Cl.sub.2 (3×20 mL). The combined filtrate (70 mL) was washed with 20% aq. NaHCO.sub.3 (40 mL), water (30 mL), and brine (20 mL). The organic phase was separated, dried over MgSO.sub.4, and concentrated in vacuo. The residue was purified by column chromatography on silica gel (ethyl acetate-toluene gradient elution) to afford the title compound (0.461 g, 86%) as a white foam. Analytical data for 75: Rf=0.30 (ethyl acetate/toluene, 0.5/9.5, v/v); [c]D.sub.21=+52.7 (c=1.0, CHCl.sub.3); .sup.1H NMR (500 MHz, CDCl.sub.3): δ 7.16-8.09 (m, 30H, H—Ar), 5.60 (dd, J.sub.2,3=3.1 Hz, 1H, H-2.sup.E), 4.98 (d, J.sub.1,2=1.8 Hz, 1H, H-1.sup.D), 4.92 (d, J.sub.1,2=1.8 Hz, 1H, H-1.sup.E), 4.88 (d, J.sub.1,2=2.0 Hz, 1H, H-1.sup.C), 4.86 (d, J.sub.1,2=2.0 Hz, 1H, H-1.sup.B), 1.23-1.29 (m, 9H, H-6.sup.B, H-6.sup.C H-6.sup.D), 1.19 (d, J.sub.5,6=6.2 Hz, 3H, H-6.sup.E), 1.15 ppm (d, J.sub.5,6=6.2 Hz, 3H, H-6.sup.A); .sup.13C NMR (126 MHz, CDCl.sub.3): δ 173.9, 165.3, 137.4, 137.3, 137.1 (×2), 133.3, 129.9, 129.8, 128.7, 128.6 (×3), 128.5 (×2), 128.4 (×2), 128.3 (×3), 128.2 (×2), 128.1 (×3), 127.9 (×2), 100.4, 100.2, 100.1, 99.2, 98.6, 77.4, 76.6, 75.3, 74.1, 74.0, 73.6, 72.2 (×2), 72.1 (×2), 71.3, 67.8 (×2), 67.7 (×2), 67.5, 67.1, 64.4, 64.3, 64.2, 64.1 (×2), 51.5, 33.9, 29.0, 25.7, 24.7, 18.6, 18.5 (×2) ppm; HRMS (ESI): m/z calcd for C.sub.79H.sub.93N.sub.15O.sub.19Na [M+Na].sup.+: 1578.6664. found: 1578.6667.

5′-Methoxycarbonylpentyl 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (76)

(162) Sodium methoxide (0.8 mL, 0.5 M solution) was added to a solution of 75 (0.450 g, 0.289 mmol) in CH.sub.3OH (10 mL) until pH ˜9 and the resulting mixture was stirred under argon for 4 h at room temperature. Then the reaction mixture was neutralized with Amberlite IR 120 (H.sup.+) ion exchange resin, the resin was filtered off and rinsed successively with CH.sub.3OH. The combined filtrate was concentrated in vacuo and purified by column chromatography on silica gel (ethyl acetate-toluene gradient elution) to afford the title compound (0.395 g, 94%) as oil. Analytical data for 76: Rf=0.40 (ethyl acetate/toluene, 1/9, v/v); [α].sub.D.sup.21=+81.2 (c=1.0, CHCl.sub.3); .sup.1H NMR (600 MHz, CDCl.sub.3): δ 7.28-7.41 (m, 25H, H—Ar), 4.97 (d, J.sub.1,2=1.1 Hz, 1H, H-1.sup.E), 4.96 (d, J.sub.1,2=1.5 Hz, 1H, H-1.sup.D), 4.87 (d, J.sub.1,2=1.3 Hz, 1H, H-1.sup.C), 4.85 (d, J.sub.1,2=1.3 Hz, 1H, H-1.sup.B), 1.14-1.26 ppm (m, 15H, H-6.sup.A, H-6.sup.B, H-6.sup.C, H-6.sup.D, H-6.sup.E); .sup.13C NMR (126 MHz, CDCl.sub.3): δ 174.0, 137.3 (×2), 137.2, 137.1 (×2), 128.6 (×4), 128.4 (×2), 128.3 (×3), 128.2 (×2), 128.1 (×2), 100.5, 100.4, 100.2 (×2), 98.6, 77.7, 77.4, 76.6, 76.5, 74.0, 73.6, 73.5, 73.3, 72.2 (×2), 72.1 (×2), 67.8, 67.7, 67.5, 67.3, 67.1 (×2), 64.4, 64.2, 63.8, 51.5, 33.9, 29.0, 25.7, 24.7, 18.6 (×2), 18.5 (×2), 18.3 ppm; HRMS (ESI): m/z calcd for C.sub.72H.sub.89N.sub.5O.sub.18Na [M+Na].sup.+: 1474.6402. found: 1474.6406.

5′-Methoxycarbonylpentyl 4-azido-2,3-di-O-benzoyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (77)

(163) A mixture of glycosyl donor 53 (0.158 g, 0.292 mmol), glycosyl acceptor 76 (0.3 86 g, 0.266 mmol) and freshly activated molecular sieves (3 Å, 0.5 g) in CH.sub.2Cl.sub.2 (5 mL) was stirred under argon for 5 h at room temperature. TMSOTf (11 μL, 0.058 mmol) was added and the resulting mixture was stirred for an additional hour. Then Et.sub.3N (1 mL) was added, the solid was filtered off and the residue was rinsed with CH.sub.2Cl.sub.2 (3×20 mL). The combined filtrate (80 mL) was washed with 20% aq. NaHCO.sub.3 (50 mL), water (30 mL), and brine (20 mL). The organic phase was separated, dried over MgSO.sub.4, and concentrated in vacuo. The residue was purified by column chromatography on silica gel (ethyl acetate-toluene gradient elution) to afford the title compound (0.487 g, 90%) as a white foam. Analytical data for 77: Rf=0.70 (ethyl acetate/toluene, 1/9, v/v); [α].sub.D.sup.21=+31.7 (c=1.4, CHCl.sub.3); .sup.1H NMR (600 MHz, CDCl.sub.3): δ 7.13-8.03 (m, 35H, H—Ar), 5.70 (dd, J.sub.2,3=3.3 Hz, 1H, H-2.sup.F), 5.59 (dd, J.sub.3,4=10.2 Hz, 1H, H-3.sup.F), 5.02-5.03 (m, 2H, H-1.sup.E, H-1), 4.90 (dd, J.sub.1,2=1.8 Hz, 1H, H-1.sup.D), 4.88 (dd, J.sub.1,2=1.8 Hz, 1H, H-1.sup.C), 4.86 (dd, J.sub.1,2=1.8 Hz, 1H, H-1.sup.B), 1.14-1.31 ppm (m, 18H, H-6.sup.A, H-6.sup.B, H-6.sup.C, H-6.sup.D, H-6.sup.E, H-6.sup.F); .sup.13C NMR (126 MHz, CDCl.sub.3): δ 174.0, 165.2, 164.9, 137.4, 137.3, 137.1 (×3), 133.4, 133.2, 129.8, 129.7, 129.5, 129.3, 129.0, 128.7, 128.6 (×3), 128.5 (×2), 128.4 (×3), 128.3 (×2), 128.2, 128.1 (×2), 127.9 (×3), 100.4, 100.3, 100.1, 100.0, 98.9, 98.6, 77.4, 77.1, 76.6, 76.5, 74.0, 73.8, 73.6, 73.5, 73.0, 72.3 (×2), 72.2 (×2), 72.1, 70.9, 69.4, 68.1, 67.9, 67.8 (×2), 67.6, 67.5, 67.1, 64.4, 64.3, 64.2 (×2), 63.9, 63.4, 51.5, 33.9, 29.0, 25.7, 24.7, 18.6 (×2), 18.5 (×2), 18.4 ppm; HRMS (ESI): m/z calcd for C.sub.92H.sub.106N.sub.18O.sub.23Na [M+Na].sup.+: 1853.7570. found: 1853.7550.

5′-Methoxycarbonylpentyl 4-azido-3-O-benzoyl-2-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (78)

(164) A mixture of glycosyl donor 58 (0.228 g, 0.433 mmol), glycosyl acceptor 74 (0.366 g, 0.394 mmol) and freshly activated molecular sieves (3 Å, 0.500 g) in PhMe (10 mL) was stirred under argon for 2 h at room temperature. Then it was heated to 95° C. and TMSOTf (16 μL, 0.087 mmol) was added, and the mixture was stirred for an additional 60 min. Then Et.sub.3N (1 mL) was added, the solid was filtered off and the residue was rinsed with CH.sub.2Cl.sub.2 (3×30 mL). The combined filtrate (100 mL) was washed with 20% aq. NaHCO.sub.3 (50 mL), water (30 mL), and brine (30 mL). The organic phase was separated, dried over MgSO.sub.4, and concentrated in vacuo. The residue was purified by column chromatography on silica gel (ethyl acetate-toluene gradient elution) to afford the title compound (0.444 g, 87%) as a white foam. Analytical data for 78: Rf=0.50 (ethyl acetate/toluene, 0.5/9.5, v/v); [α].sub.D.sup.21=+42.1 (c=1.0, CHCl.sub.3); .sup.1H NMR (600 MHz, CDCl.sub.3): δ 7.03-8.05 (m, 25H, H—Ar), 5.29 (dd, J.sub.3,4=10.3 Hz, 1H, H-3.sup.D), 5.07 (d, J.sub.1,2=1.6 Hz, 1H, H-1.sup.D), 4.96 (d, J.sub.1,2=1.8 Hz, 1H, H-1.sup.C), 4.91 (d, J.sub.1,2=1.8 Hz, 1H, H-1.sup.B), 1.22-1.29 ppm (m, 12H, H-6.sup.A, H-6.sup.B, H-6.sup.C, H-6.sup.D); .sup.13C NMR (126 MHz, CDCl.sub.3): δ 174.0, 165.4, 137.4, 137.3 (×2), 137.1, 133.3, 129.8, 129.5, 128.7, 128.6 (×2), 128.4, 128.3 (×2), 128.2 (×2), 128.1, 127.8, 127.6, 100.6, 100.1, 98.8, 98.6, 77.9, 77.4, 76.9, 74.4, 74.2, 72.9, 72.8, 72.7, 72.6, 72.5, 72.3, 72.2, 68.1, 67.8, 67.7, 67.5, 67.1, 64.4, 64.2, 64.1, 63.1, 51.5, 33.9, 29.0, 25.7, 24.6, 18.6 (×2), 18.4, 18.3 ppm; HRMS (ESI): m/z calcd for C.sub.66H.sub.78N.sub.12O.sub.16Na [M+Na].sup.+: 1317.5551. found: 1317.5549.

5′-Methoxycarbonylpentyl 4-azido-2-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (79)

(165) Sodium methoxide (0.8 mL, 0.5 M solution) was added to a solution of 78 (0.434 g, 0.335 mmol) in CH.sub.3OH (10 mL) until pH ˜9 and the resulting mixture was stirred under argon for 4 h at room temperature. Then the reaction mixture was neutralized with Amberlite IR 120 (H.sup.+) ion exchange resin, the resin was filtered off and rinsed successively with CH.sub.3OH. The combined filtrate was concentrated in vacuo and purified by column chromatography on silica gel (ethyl acetate-toluene gradient elution) to afford the title compound (0.356 g, 89%) as a white foam. Analytical data for 79: Rf=0.50 (ethyl acetate/toluene, 1/9, v/v); [α].sub.D.sup.21=+65.6 (c=1.3, CHCl.sub.3); .sup.1H NMR (500 MHz, CDCl.sub.3): δ 7.13-7.42 (m, 20H, H—Ar), 5.10 (d, J.sub.1,2=0.9 Hz, 1H, H-1.sup.D), 4.93 (d, J.sub.1,2=1.7 Hz, 1H, H-1.sup.C), 4.90 (d, J.sub.1,2=1.8 Hz, 1H, H-1.sup.B), 1.19-1.29 ppm (m, 12H, H-6.sup.A, H-6.sup.B, H-6.sup.C, H-6.sup.D); .sup.13C NMR (126 MHz, CDCl.sub.3): δ 174.0, 137.3 (×2), 137.2, 137.1, 128.7, 128.6 (×2), 128.4, 128.3 (×2), 128.2, 128.1 (×2), 100.5, 100.2, 98.6, 97.8, 77.8, 77.5, 76.9, 76.5, 74.1, 73.1, 72.7, 72.5, 72.3, 72.2, 70.0, 67.9, 67.8, 67.5, 67.3, 67.1, 66.5, 64.5, 64.4, 64.3, 51.5, 33.9, 29.1, 25.7, 24.7, 18.7, 18.6, 18.4, 18.3 ppm; HRMS (ESI): m/z calcd for C.sub.59H.sub.74N.sub.12O.sub.15Na [M+Na].sup.+: 1213.5289. found: 1213.5284.

5′-Methoxycarbonylpentyl 4-azido-2,3-di-O-benzoyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→3) 4-azido-2-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (80)

(166) A mixture of glycosyl donor 63 (0.220 g, 0.313 mmol), glycosyl acceptor 79 (0.339 g, 0.285 mmol) and freshly activated molecular sieves (3 Å, 0.5 g) in CH.sub.2Cl.sub.2 (10 mL) was stirred under argon for 4 h at room temperature. MeOTf (260 μL, 2.28 mmol) was added and continued stirring for additional 48 h. Then Et.sub.3N (1 mL) was added, the solid was filtered off and the residue was rinsed with CH.sub.2Cl.sub.2 (3×30 mL). The combined filtrate (100 mL) was washed with 20% aq. NaHCO.sub.3 (40 mL), water (40 mL), and brine (20 mL). The organic phase was separated, dried over MgSO.sub.4, and concentrated in vacuo. The residue was purified by column chromatography on silica gel (ethyl acetate-toluene gradient elution) to afford the title compound (0.479 g, 92%) as a white foam. Analytical data for 80: Rf=0.60 (ethyl acetate/toluene, 0.5/9.5, v/v); [c]D.sub.21=+15.3 (c=1.0, CHCl.sub.3); .sup.1H NMR (600 MHz, CDCl.sub.3): δ 7.06-8.00 (m, 35H, H—Ar), 5.71 (dd, J.sub.2,3=3.3 Hz, 1H, H-2.sup.F), 5.58 (dd, J.sub.3,4=10.3 Hz, 1H, H-3.sup.F), 5.09 (d, J.sub.1,2=1.2 Hz, 1H, H-1.sup.E), 5.05-5.07 (m, 2H, H-1.sup.D, H-1.sup.F), 4.95 (d, J.sub.1,2=1.5 Hz, 1H, H-1.sup.C), 4.90 (d, J.sub.1,2=1.7 Hz, 1H, H-1.sup.B), 1.20-1.29 ppm (m, 15H, H-6.sup.A H-6.sup.B, H-6.sup.C, H-6.sup.D, H-6.sup.E); .sup.13C NMR (126 MHz, CDCl.sub.3): δ 174.0, 165.2, 164.9, 137.5, 137.4, 137.3, 137.2, 137.1, 133.4, 133.3, 129.8 (×2), 129.5, 129.4, 129.0, 128.7, 128.6 (×2), 128.5 (×3), 128.4 (×2), 128.3 (×2), 128.2 (×2), 128.1, 127.9, 127.8, 127.7, 127.6, 100.9, 100.5, 100.2, 99.2, 98.6, 98.2, 77.9, 77.7, 77.5, 76.9, 76.4, 74.1, 73.6, 73.1, 72.9, 72.5, 72.4, 72.2 (×2), 72.0, 71.0, 69.4, 68.2, 68.1, 67.9, 67.8, 67.7, 67.5, 67.1, 64.7, 64.5, 64.4, 64.3, 63.6, 63.5, 51.5, 33.9, 29.1, 25.7, 24.7, 18.6 (×3), 18.5 (×2), 18.4 ppm; HRMS (ESI): m/z calcd for C.sub.92H.sub.110N.sub.19O.sub.23 [M+NH.sub.4].sup.+: 1848.8016. found: 1848.8005.

5′-Methoxycarbonylpentyl 4-azido-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (77a)

(167) Sodium methoxide (0.8 mL, 0.5 M solution) was added to a solution of 77 (0.483 g, 0.264 mmol) in CH.sub.3OH (12 mL) until pH ˜9 and the resulting mixture was stirred under argon for 6 h at room temperature. Then the reaction mixture was neutralized with Amberlite IR 120 (H.sup.+) ion exchange resin, the resin was filtered off and rinsed successively with CH.sub.3OH. The combined filtrate was concentrated in vacuo and purified by column chromatography on silica gel (ethyl acetate-toluene gradient elution) to afford the title compound (0.375 g, 87%) as oil. Analytical data for 77a: Rf=0.30 (ethyl acetate/toluene, 1.5/8.5, v/v); [α].sub.D.sup.21=+101.4 (c=1.1, CHCl.sub.3); .sup.1H NMR (500 MHz, CDCl.sub.3): δ 7.28-7.40 (m, 25H, H—Ar), 4.98 (d, J.sub.1,2=1.5 Hz, 1H, H-1.sup.F), 4.90 (d, J.sub.1,2=1.5 Hz, 1H, H-1.sup.E), 4.89 (d, J.sub.1,2=1.5 Hz, 1H, H-1.sup.D), 4.86 (d, J.sub.1,2=2.0 Hz, 1H, H-1.sup.C), 4.85 (d, J.sub.1,2=2.0 Hz, 1H, H-1.sup.B), 1.13-1.26 ppm (m, 18H, H-6.sup.A H-6.sup.B, H-6.sup.C, H-6.sup.D, H-6.sup.E, H-6.sup.F); .sup.13C NMR (126 MHz, CDCl.sub.3): δ 174.0, 137.4 (×2), 137.2 (×3), 128.7 (×2), 128.6 (×2), 128.4 (×2), 128.3 (×4), 128.1 (×2), 100.7, 100.4, 100.2 (×2), 100.1, 98.6, 77.5, 76.9, 76.6 (×2), 74.1, 73.6, 73.5, 73.3, 73.2, 72.3 (×2), 72.2 (×2), 70.2, 70.0, 67.9, 67.8 (×2), 67.5, 67.4, 67.1, 65.8, 64.4, 64.3, 64.2, 51.5, 33.9, 29.1, 25.7, 24.7, 18.6 (×2), 18.5 (×3), 18.2 ppm; HRMS (ESI): m/z calcd for C.sub.78H.sub.98N.sub.18O.sub.21Na [M+Na].sup.+: 1645.7046. found: 1645.7043.

5′-Methoxycarbonylpentyl 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranoside (48)

(168) To a stirred solution of 77a (0.130 g, 0.080 mmol), in pyridine (5 mL) and water (2 mL) mixture, H.sub.2S was bubbled for 0.5 h at 40° C., and stirring was continued for 16 h. Then argon was bubbled through the solution for 10 min, solvents were removed in vacuo, and the residue was co-evaporated with toluene (3×10 mL) and dried. The high resolution mass spectrometry analysis showed completion of reaction to corresponding amine compound 77b and no products arising from incomplete reduction. HRMS (ESI): m/z calcd for C.sub.78H.sub.111N.sub.6O.sub.21 [M+H].sup.+: 1467.7797. found: 1467.7795. This crude material was directly used for formylation.

(169) Compound 77b in CH.sub.3OH (5 mL) at −20° C. was added a freshly prepared formic anhydride (Angew (1979) Chem. Int. Ed. 18, 614) (5 mL, ethereal solution) and stirred for 3 h, then slowly allowed to warm to room temperature. Then solvents were evaporated and the residue was passed through column chromatography on silica gel (methanol-dichloromethane gradient elution) to afford disaccharide 77c. HRMS (ESI): m/z calcd for C.sub.84H.sub.111N.sub.6O.sub.27Na [M+H].sup.+: 1635.7492. found: 1635.7485.

(170) Compound 77c was dissolved in CH.sub.3OH/H.sub.2O (2:1, 5 mL), Pd(OH).sub.2 on carbon (20%, 0.050 g) was added. Then it was stirred under a pressure of hydrogen gas at room temperature for 16 h. After filtration through celite pad and washed with CH.sub.3OH (3×10 mL), and solvents were removed in vacuo. The residue was purified by column chromatography on silica gel (methanol-dichloromethane gradient elution) to afford the title compound (0.052 g, 55%, over 3 steps) as a white foam. Analytical data for 48: Rf=0.20 (CH.sub.3OH/CH.sub.2Cl.sub.2, 2/3, v/v); .sup.1H NMR (500 MHz, D.sub.2O): δ 8.17-8.19 (Z) and 7.99-8.02 (E) (m, 6H, NCHO), 4.84-5.20 (m, 6H, 6×H-1), 1.16-1.27 ppm (m, 18H, 6×H-6); .sup.13C NMR (126 MHz, D.sub.2O): 6178.5, 168.8, 165.9, 103.0, 102.9, 101.6, 101.5 (×2), 99.2, 78.6, 78.3, 78.1 (×2), 78.0 (×2), 77.9, 69.9 (×2), 69.2 (×2), 69.0, 68.9 (×2), 68.8, 68.7, 68.6 (×2), 68.5 (×3), 57.9, 57.7, 53.1, 53.0, 52.9, 52.8 (×2), 52.7, 49.9, 34.6, 29.0, 25.8, 24.9, 17.9 (×2), 17.8, 17.7 (×2), 17.6 (×2) ppm; HRMS (ESI): m/z calcd for C.sub.49H.sub.80N.sub.6O.sub.27Na [M+Na].sup.+: 1207.4964. found: 1207.4941.

(2′-Aminoethylamido)carbonylpentyl 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranoside (85)

(171) A solution of 48 (0.034 g, 0.029 mmol) in freshly distilled 1,2-diaminoethane (0.5 mL) was stirred at 65° C. for 48 h. Then excess reagent was removed in vacuo, and the residue was co-evaporated with CH.sub.3OH (3×10 mL) and dried. The residue was purified by reversed phase HPLC on a C18 column with a gradient of water-acetonitrile and lyophilized, to give the title compound (0.034 g, 97%) as a white foam. Analytical data for 85: Rf=0.20 (CH.sub.3OH); .sup.1H NMR (500 MHz, D.sub.2O): δ 8.16-8.19 (Z) and 7.98-8.02 (E) (m, 6H, NCHO), 4.81-5.19 (m, 6H, 6×H-1), 1.14-1.26 ppm (m, 18H, 6×H-6); .sup.13C NMR (126 MHz, D.sub.2O): 6177.4, 177.1, 167.9, 167.8, 164.9, 164.6, 102.4, 102.1, 102.0 (×2), 100.8, 100.7, 100.6 (×2), 98.6, 98.3 (×2), 77.6, 77.4, 77.3, 77.2, 77.1, 77.0, 76.8, 70.4, 70.3, 70.1, 70.0, 69.4, 69.2, 69.0 (×2), 68.3 (×2), 68.0 (×2), 67.9 (×2), 67.8 (×2), 67.7 (×2), 67.6 (×2), 67.5 (×2), 67.0, 57.0, 56.9, 56.7, 53.9, 53.8, 53.6, 52.0, 51.9 (×2), 51.8 (×2), 51.7, 40.9, 40.4, 40.0, 39.7, 35.7, 28.2, 25.0 (×2), 24.9, 17.1 (×2), 17.0 (×2), 16.9 (×3), 16.8, 16.7 ppm; HRMS (ESI): m/z calcd for C.sub.50H.sub.84N.sub.8O.sub.26Na [M+Na].sup.+: 1235.5389. found: 1235.5384.

1-[(2′-Aminoethylamido)carbonylpentyl 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranoside]-2-butoxycyclobutene-3,4-dione (91)

(172) To a stirred solution of 85 (0.0142 g, 0.012 mmol) in water (0.5 mL) and EtOH (0.4 mL), a solution of 3,4-dibutoxy-3-cyclobutene-1,2-dione (20% in ethanol, 55 μL) was added and the pH was adjusted to 8 by careful addition of aq.NaHCO.sub.3 (1%) solution. After 0.5 h, TLC showed the reaction was complete; the reaction mixture was neutralized using CH.sub.3COOH (10%) and concentrated in vacuo. The residue was purified by reversed phase HPLC on a C18 column with a gradient of water-acetonitrile and lyophilized, to give the title compound (0.014 g, 88%) as a white foam. Analytical data for 91: Rf=0.40 (CH.sub.3OH/CH.sub.2Cl.sub.2, 1/1, v/v); .sup.1H NMR (700 MHz, D.sub.2O): δ 8.20-8.23 (Z) and 8.02-8.07 (E) (m, 6H, NCHO), 4.88-5.34 (m, 6H, 6×H-1), 1.17-1.35 (m, 20H, —CH.sub.2d—, 6×H-6), 0.88-1.00 ppm (m, 3H, —CH.sub.31); .sup.13C NMR (126 MHz, D.sub.2O): δ 189.8, 189.6, 184.3, 178.5, 178.1, 177.9, 174.8, 174.7, 168.8, 165.9, 102.9 (×2), 101.5 (×3), 99.2 (×2), 78.5, 78.1 (×3), 78.0 (×2), 75.4, 75.3, 69.9, 69.2, 69.0, 68.9, 68.7, 68.5, 57.9, 57.7, 53.0, 52.9 (×2), 52.6, 45.2, 45.0, 40.3, 40.2, 36.7, 32.4, 32.3, 29.2, 26.1, 26.0, 25.9 (×2), 25.9, 19.2, 19.1, 19.0, 17.9 (×2), 17.8 (×2), 17.7 (×2), 13.9 ppm; HRMS (ESI): m/z calcd for C.sub.58H.sub.92NsO.sub.29Na [M+Na].sup.+: 1387.5862. found: 1387.5856.

5′-Methoxycarbonylpentyl 4-azido-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→3) 4-azido-2-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (80a)

(173) Sodium methoxide (0.8 mL, 0.5 M solution) was added to a solution of 80 (0.413 g, 0.225 mmol) in CH.sub.3OH (12 mL) until pH ˜9 and the resulting mixture was stirred under argon for 6 h at room temperature. Then the reaction mixture was neutralized with Amberlite IR 120 (H.sup.+) ion exchange resin, the resin was filtered off and rinsed successively with CH.sub.3OH. The combined filtrate was concentrated in vacuo and purified by column chromatography on silica gel (ethyl acetate-toluene gradient elution) to afford the title compound (0.334 g, 91%) as a white foam. Analytical data for 80a: Rf=0.40 (ethyl acetate/toluene, 1/9, v/v); [α].sub.D.sup.21=+61.0 (c=1.0, CHCl.sub.3); .sup.1H NMR (500 MHz, CDCl.sub.3): δ 7.14-7.38 (m, 25H, H—Ar), 5.03-5.05 (br. s., 2H, H-1.sup.D, H-1.sup.E), 4.94 (d, J.sub.1,2=1.5 Hz, 1H, H-1.sup.C), 4.91 (d, J.sub.1,2=1.2 Hz, 1H, H-1.sup.B), 4.90 (d, J.sub.1,2=1.5 Hz, 1H, H-1.sup.F), 1.15-1.35 ppm (m, 20H, —CH.sub.2d—, H-6.sup.A, H-6.sup.B, H-6.sup.C, H-6.sup.D, H-6.sup.E, H-6.sup.F); .sup.13C NMR (126 MHz, CDCl.sub.3): δ 174.0, 137.5, 137.4, 137.3, 137.2, 137.1, 128.7, 128.6 (×3), 128.4 (×2), 128.3 (×2), 128.2 (×2), 128.1 (×2), 128.0 (×2), 127.9, 127.5, 101.0, 100.9, 100.4, 100.2, 98.6, 98.1, 78.0, 77.6, 77.5 (×2), 76.9, 76.4, 74.0, 73.3, 73.1, 72.9, 72.5, 72.4, 72.2, 72.1, 71.9, 70.2, 69.9, 68.1, 67.9 (×2), 67.8, 67.5 (×2), 67.1, 65.8, 64.6, 64.5, 64.4, 64.3, 64.0, 51.5, 33.9, 29.1, 25.7, 24.7, 18.6 (×3), 18.5, 18.4, 18.3 ppm; HRMS (ESI): m/z calcd for C.sub.78H.sub.98N.sub.18O.sub.21Na [M+Na].sup.+: 1645.7046. found: 1645.7035.

5′-Methoxycarbonylpentyl 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→3) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranoside (49)

(174) To a stirred solution of 80a (0.150 g, 0.092 mmol), in pyridine (5 mL) and water (2 mL) mixture, H.sub.2S was bubbled for 0.5 h at 40° C., and stirring was continued for 16 h. Then argon was bubbled through the solution for 10 min, solvents were removed in vacuo, and the residue was co-evaporated with toluene (3×10 mL) and dried. The high resolution mass spectrometry analysis showed completion of reaction to corresponding amine compound 80b and no products arising from incomplete reduction. HRMS (ESI): m/z calcd for C.sub.78H.sub.111N.sub.6O.sub.21 [M+H].sup.+: 1467.7797. found: 1467.7781. This crude material was directly used for formylation.

(175) Compound 80b in CH.sub.3OH (5 mL) at −20° C. was added a freshly prepared formic anhydride.sup.[22] (5 mL, ethereal solution) and stirred for 3 h, then slowly allowed to warm to room temperature. Then solvents were evaporated and the residue was passed through column chromatography on silica gel (methanol-dichloromethane gradient elution) to afford disaccharide 80c. HRMS (ESI): m/z calcd for C.sub.84H.sub.110N.sub.6O.sub.27Na [M+Na].sup.+: 1657.7311. found: 1657.7314.

(176) Compound 80c was dissolved in CH.sub.3OH/H.sub.2O (2:1, 5 mL), Pd(OH).sub.2 on carbon (20%, 0.050 g) was added. Then it was stirred under a pressure of hydrogen gas at room temperature for 16 h. After filtration through celite pad and washed with CH.sub.3OH (3×10 mL), and solvents were removed in vacuo. The residue was purified by column chromatography on silica gel (methanol-dichloromethane gradient elution) to afford the title compound (0.066 g, 60%, over 3 steps) as a white foam. Analytical data for 49: Rf=0.30 (CH.sub.3OH/CH.sub.2Cl.sub.2, 1/1, v/v); .sup.1H NMR (700 MHz, D.sub.2O): δ 8.20-8.25 (Z) and 8.02-8.06 (E) (m, 6H, NCHO), 4.89-5.23 (m, 6H, 6×H-1), 1.19-1.31 ppm (m, 18H, 6×H-6); .sup.13C NMR (176 MHz, D.sub.2O): δ 178.4, 168.6, 165.7 (×2), 165.4 (×2), 103.1, 102.4, 101.5 (×2), 101.3, 99.1 (×2), 78.8, 78.4, 78.2, 78.0, 77.9, 77.6, 77.2, 69.7 (×2), 69.6 (×2), 69.5, 69.1, 69.0, 68.8, 68.7 (×2), 68.6, 68.5, 68.4 (×2), 68.3, 68.2, 67.9, 57.8, 57.7, 57.6, 52.9, 52.8, 52.7, 52.6, 52.5, 51.7, 34.4, 28.9, 25.7, 24.8, 18.0, 17.9, 17.8 (×2), 17.7 (×2), 17.6 (×3), 17.5 ppm; HRMS (ESI): m/z calcd for C.sub.49H.sub.80N.sub.6O.sub.27Na [M+Na].sup.+: 1207.4964. found: 1207.4963.

(2′-Aminoethylamido)carbonylpentyl 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→3) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranoside (86)

(177) A solution of 49 (0.041 g, 0.035 mmol) in freshly distilled 1,2-diaminoethane (0.5 mL) was stirred at 65° C. for 48 h. Then excess reagent was removed in vacuo, and the residue was co-evaporated with CH.sub.3OH (3×10 mL) and dried. The residue was purified by reversed phase HPLC on a C18 column with a gradient of water-acetonitrile and lyophilized, to give the title compound (0.039 g, 93%) as a white foam. Analytical data for 86: R.sub.f=0.20 (CH.sub.3OH); .sup.1H NMR (500 MHz, D.sub.2O): δ 8.25-8.32 (Z) and 8.08-8.14 (E) (m, 6H, NCHO), 4.92-5.30 (m, 6H, 6×H-1), 1.23-1.39 ppm (m, 18H, 6×H-6); .sup.13C NMR (126 MHz, D.sub.2O): δ 178.4, 178.1, 168.8 (×2), 165.9, 165.6, 103.3 (×2), 102.6, 101.9, 101.8, 101.6, 101.5, 99.3, 99.2, 78.9, 78.6, 78.5, 78.1, 77.8, 77.4, 71.0, 69.9 (×2), 69.7 (×2), 69.6, 69.4, 69.2, 69.0, 69.0, 68.9, 68.8, 68.6 (×2), 68.5 (×2), 68.4, 68.0, 57.9 (×2), 57.8, 57.7, 56.4, 53.0, 52.8 (×2), 52.7, 51.8, 41.4, 41.1, 40.6, 36.6, 29.1, 26.0, 25.9 (×2), 18.1 (×3), 18.0, 17.9, 17.8, 17.7, 17.6 ppm; HRMS (ESI): m/z calcd for C.sub.50H.sub.85N.sub.8O.sub.26 [M+H].sup.+: 1213.5570. found: 1213.5564.

1-[(2′-Aminoethylamido)carbonylpentyl 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→3) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranoside]-2-butoxycyclobutene-3,4-dione (92)

(178) To a stirred solution of 86 (0.014 g, 0.011 mmol) in water (0.8 mL) and EtOH (0.6 mL), a solution of 3,4-dibutoxy-3-cyclobutene-1,2-dione (20% in ethanol, 55 μL) was added and the pH was adjusted to 8 by careful addition of aq.NaHCO.sub.3 (1%) solution. After 0.5 h, TLC showed the reaction was complete; the reaction mixture was neutralized using CH.sub.3COOH (10%) and concentrated in vacuo. The residue was purified by reversed phase HPLC on a C18 column with a gradient of water-acetonitrile and lyophilized, to give the title compound (0.012 g, 76%) as a white foam. Analytical data for 92: Rf=0.40 (CH.sub.3OH/CH.sub.2Cl.sub.2, 1/1, v/v); .sup.1H NMR (700 MHz, D.sub.2O): δ 8.20-8.25 (Z) and 8.02-8.07 (E) (m, 6H, NCHO), 4.85-5.21 (m, 6H, 6×H-1), 1.18-1.35 (m, 20H, —CH.sub.2d—, 6×H-6), 0.89-0.97 ppm (m, 3H, —CH.sub.31); .sup.13C NMR (126 MHz, D.sub.2O): δ 189.8, 189.7, 184.3, 178.5, 178.1, 177.9 (×2), 174.8, 174.7, 168.8, 165.9, 165.6, 103.3, 102.6, 101.6, 101.5, 99.2, 78.9, 78.5, 78.2, 77.8, 77.3, 75.4, 75.3, 70.8, 69.9, 69.7, 69.2, 69.0, 68.8, 68.7, 68.6, 68.5, 68.4, 57.9, 57.7, 53.0, 52.8, 52.7, 51.8, 45.2, 45.0, 40.3, 40.2, 36.7, 32.4, 29.2, 26.1, 26.0, 25.9 (×2), 19.1, 19.0, 18.1, 18.0, 17.9 (×2), 17.8, 17.7, 13.9 ppm; HRMS (ESI): m/z calcd for C.sub.58H.sub.92N.sub.8O.sub.29Na [M+Na].sup.+: 1387.5862. found: 1387.5864.

(179) Conjugation of the Pentasaccharide Squarate 40 to BSA (42)

(180) BSA (11.7 mg, 0.17 mol) and squarate 40 (4.2 mg, 3.5 μmol) were dissolved in 0.5 M borate buffer pH 9 (350 μL) and stirred gently at room temperature for 3 days. Then the reaction mixture was diluted with water and dialyzed against deionized water (3×2 L) at 4° C., spinned and lyophilized. The product of conjugation was obtained as a white solid (12.5 mg, 84%): MALDI-TOF-MS indicated the conjugate 42 had an average of 16.4 pentasaccharides per BSA.

(181) Conjugation of the Nonasaccharide Squarate 41 to BSA (43)

(182) The conjugation of BSA (5.5 mg, 0.08 μmol) and squarate 41 (3.2 mg, 1.6 μmol) in borate buffer (220 μl) was performed as described for 42. After the dialysis (5×2 L) and lyophilization the conjugate 43 was obtained as a white solid (6 mg, 74%). As calculated from MALDI-TOF-MS spectrum the average number of nonasaccharides per BSA was 16.8.

(183) Conjugation of Squarate Derivative 87 to BSA (93).

(184) BSA (30 mg, 0.451 μmol) and disaccharide squarate 87 (4.5 mg, 6.77 μmol) were dissolved in 0.5 M borate buffer pH 9 (600 μL) and stirred gently at room temperature for 3 days. Then the reaction mixture was diluted with Mili-Q water, filtered through milipore filtration tube (10,000 MWCO, 4×10 mL), lyophilized and the BSA-conjugate 93 was obtained as a white foam (30.4 mg, 89%). The MALDI-TOF mass spectrometry analysis indicated the conjugate 93 had an average of 15.2 disaccharides per BSA.

(185) Conjugation of Squarate Derivative 88 to BSA (94).

(186) BSA (30 mg, 0.451 mol) and trisaccharide squarate 88 (5.7 mg, 6.74 μmol) were dissolved in 0.5 M borate buffer pH 9 (700 μL) and stirred gently at room temperature for 3 days. Then the reaction mixture was diluted with Mili-Q water, filtered through milipore filtration tube (10,000 MWCO, 4×10 mL), lyophilized and the BSA-conjugate 94 was obtained as a white foam (32.3 mg, 91%). The MALDI-TOF mass spectrometry analysis indicated the conjugate 94 had an average of 15.9 trisaccharides per BSA.

(187) Conjugation of Squarate Derivative 89 to BSA (95).

(188) BSA (32.5 mg, 0.489 μmol) and trisaccharide squarate 89 (6.2 mg, 7.34 μmol) were dissolved in 0.5 M borate buffer pH 9 (700 μL) and stirred gently at room temperature for 3 days. Then the reaction mixture was diluted with Mili-Q water, filtered through milipore filtration tube (10,000 MWCO, 4×10 mL), lyophilized and the BSA-conjugate 95 was obtained as a white foam (33.5 mg, 87%). The MALDI-TOF mass spectrometry analysis indicated the conjugate 95 had an average of 15.7 trisaccharides per BSA.

(189) Conjugation of Squarate Derivative 90 to BSA (96).

(190) BSA (11 mg, 0.165 μmol) and tetrasaccharide squarate 90 (2.5 mg, 2.45 μmol) were dissolved in 0.5 M borate buffer pH 9 (400 μL) and stirred gently at room temperature for 3 days. Then the reaction mixture was diluted with Mili-Q water, filtered through milipore filtration tube (10,000 MWCO, 4×10 mL), lyophilized and the BSA-conjugate 96 was obtained as a white foam (12 mg, 92%). The MALDI-TOF mass spectrometry analysis indicated the conjugate 96 had an average of 13.4 tetrasaccharides per BSA.

(191) Conjugation of Squarate Derivative 91 to BSA (97).

(192) BSA (5 mg, 0.0752 mol) and hexasaccharide squarate 91 (1.5 mg, 1.099 μmol) were dissolved in 0.5 M borate buffer pH 9 (400 μL) and stirred gently at room temperature for 3 days. Then the reaction mixture was diluted with Mili-Q water, filtered through milipore filtration tube (10,000 MWCO, 4×10 mL), lyophilized and the BSA-conjugate 97 was obtained as a white foam (5.5 mg, 87%). The MALDI-TOF mass spectrometry analysis indicated the conjugate 97 had an average of 13.8 hexasaccharides per BSA.

(193) Conjugation of Squarate Derivative 92 to BSA (98).

(194) BSA (5 mg, 0.0752 mol) and hexasaccharide squarate 92 (1.5 mg, 1.099 mol) were dissolved in 0.5 M borate buffer pH 9 (400 μL) and stirred gently at room temperature for 2 days. Then the reaction mixture was diluted with Mili-Q water, filtered through milipore filtration tube (10,000 MWCO, 4×10 mL), lyophilized and the BSA-conjugate 98 was obtained as a white foam (6 mg, 99%). The MALDI-TOF mass spectrometry analysis indicated the conjugate 98 had an average of 10.8 hexasaccharides per BSA.

(195) Conjugation of Squarate Derivative 87 to Co-Povidone Polymer (99).

(196) High Loading.

(197) Co-povidone (6.9 mg, 5.95 μmol, 1 eq.) and disaccharide squarate 87 (2.0 mg, 2.97 μmol, 0.5 eq.) were dissolved in 0.5 M borate buffer pH 9 (500 μL) and stirred gently at room temperature for 2 days. Then, a solution of 5% aq.Ac.sub.2O (1 mL) and saturated NaHCO.sub.3 (1 mL) was added and stirred for 3 h. After that, the reaction mixture was diluted with Mili-Q water, dialyzed against deionized water (5×2 L), lyophilized to obtain the co-povidone-conjugate 99a.

(198) Low Loading.

(199) Co-povidone (5.0 mg, 4.31 μmol, 1 eq.) and disaccharide squarate 87 (0.5 mg, 0.74 μmol, 0.166 eq.) were dissolved in 0.5 M borate buffer pH 9 (500 μL) and stirred gently at room temperature for 2 days. Then, a solution of 5% aq.Ac.sub.2O (1 mL) and saturated NaHCO.sub.3 (1 mL) was added and stirred for 3 h. After that, the reaction mixture was diluted with Mili-Q water, dialyzed against deionized water (5×2 L), lyophilized to obtain the co-povidone-conjugate 99b.

(200) Conjugation of Squarate Derivative 91 to Co-Povidone Polymer (100).

(201) High Loading:

(202) Co-povidone (5.0 mg, 4.31 mol, 1 eq.) and hexasaccharide squarate 91 (2.94 mg, 2.15 μmol, 0.5 eq.) were dissolved in 0.5 M borate buffer pH 9 (500 μL) and stirred gently at room temperature for 2 days. Then, a solution of 5% aq.Ac.sub.2O (1 mL) and saturated NaHCO.sub.3 (1 mL) was added and stirred for 3 h. After that, the reaction mixture was diluted with Mili-Q water, dialyzed against deionized water (5×2 L), lyophilized to obtain the co-povidone-conjugate 100a.

(203) Low Loading:

(204) Co-povidone (5.0 mg, 4.31 μmol, 1 eq.) and hexasaccharide squarate 91 (1.0 mg, 0.73 μmol, 0.166 eq.) were dissolved in 0.5 M borate buffer pH 9 (500 μL) and stirred gently at room temperature for 2 days. Then, a solution of 5% aq.Ac.sub.2O (1 mL) and saturated NaHCO.sub.3 (1 mL) was added and stirred for 3 h. After that, the reaction mixture was diluted with Mili-Q water, dialyzed against deionized water (5×2 L), lyophilized to obtain the co-povidone-conjugate 100b.

(205) Conjugation of Squarate Derivative 91 to Tetanus Toxoid (101).

(206) Hexasaccharide squarate 91 (0.55 mg, 0.403 μmol) was added to the solution of tetanus toxoid (2 mg, 0.0133 μmol) in 0.5 M borate buffer pH 9 (1 mL) and stirred gently at room temperature for 3 days. Then the reaction mixture was washed with borate buffer, filtered through milipore filtration tube (10,000 MWCO, 4×10 mL) and the resulting tetanus toxoid-conjugate 101 was stored in PBS buffer. The MALDI-TOF mass spectrometry analysis indicated the conjugate 101 had an average of 12.6 hexasaccharides per tetanus toxoid.

(207) Conjugation of Squarate Derivative 92 to Tetanus Toxoid (102).

(208) Hexasaccharide squarate 92 (0.55 mg, 0.403 μmol) was added to the solution of tetanus toxoid (2 mg, 0.0133 μmol) in 0.5 M borate buffer pH 9 (1 mL) and stirred gently at room temperature for 2 days. Then the reaction mixture was washed with borate buffer, filtered through milipore filtration tube (10,000 MWCO, 4×10 mL) and the resulting tetanus toxoid-conjugate 102 was stored in PBS buffer. The MALDI-TOF mass spectrometry analysis indicated the conjugate 102 had an average of 6.2 hexasaccharides per tetanus toxoid.

(209) Specific M-Antigen—Proof of Concept Studies on OPS from Brucella and Y. enterocolitica O:9

(210) Monoclonal antibody binding analysis using the anti-M specific antibody BM40 (Greiser et al. (1987) Ann Inst Pasteur Microbiol 138, 549-560) against Brucella A, M and Y. enterocolitica O:9 sLPS antigens, prepared by hot-phenol extraction (Westphal et al. (1952) Z. Naturforsch. 7, 148-155) confirmed the exceptionally high specificity of the BM40 response in this case. There was no measurable binding against the Y. enterocolitica O:9 sLPS and binding to B. melitensis 16M (an M dominant strain) was tenfold greater than binding to B. abortus S99 (an A dominant strain).

(211) During further exploratory studies, the absorption methods described above (Alton et al. (1994) Techniques for the Brucellosis Laboratory, pages 53-54; INRA Editions, ISBN-10: 2738000428; Kittelberger et al. (1998) Vet. Microbial. 60, 45-57) were evaluated, using sera from cattle experimentally infected with Brucella abortus strain 544 (an A dominant strain) or Y. enterocolitica O:9. A residual anti-Brucella sLPS titre was observed in indirect ELISA in Y. enterocolitica O:9 absorbed samples in some, but not all, sera from the Brucella infected animals.

(212) The sera from the experimentally infected cattle was also evaluated by indirect ELISAs using B. melitensis 16M and Y. enterocolitica O:9 OPS antigens purified from the smooth LPS using mild acid hydrolysis and size exclusion chromatography (Meikle et al. (1989) Infect Immun 57, 2820-2828), tested to be free of lipid-A using the limulus amebocyte lysate reaction (Ding et al. (2001) Trends Biotechnol. 19, 277-281). Samples taken from the cattle (cows) 3, 7, 16, 24 and 53 weeks post infection were taken from each of the four animals experimentally infected with B. abortus strain 544 and from each of the four animals experimentally infected with Y. enterocolitica O:9. These samples have been previously described and also tested by conventional serological assays for brucellosis as described previously (McGiven et al. (2008) Journal of Immunological Methods 20, 7-15).

(213) The OPS from B. melitensis 16M was chosen in preference to OPS from a B. abortus A dominant strain due to the higher frequency of α-1,3 linkages and M epitopes, so that the extremes of the structural variation in the natural 4,6-dideoxy-4-formamido-α-D-mannopyranose homopolymers would be represented.

(214) Owing to their high solubility, the purified OPS antigens do not bind effectively to ELISA polystyrene ELISA plates by passive absorption (unless conjugated to carrier molecules containing hydrophobic regions). To enable their immobilisation to a solid phase, Carbo-BIND™ 96 well ELISA plates were used (Corning, product number 2507) which have hydrazide functional groups on the well surface. These hydrazide groups react spontaneously with aldehydes which may be generated in carbohydrates by oxidation.

(215) The OPS was oxidised at 100 g/ml with 10 mM sodium metaperiodate and 50 mM sodium acetate buffer (SAB) pH 5.5 in the dark at 4° C. for 30 minutes. After this time the antigen was diluted to between 0.5 to 0.125 g/ml in SAB pH 5.5 and 100 μl was added to the wells of a Carbo-BIND™ plate. The plate was incubated for 1 hour at 37° C. and then washed with 4 times 200 μl of phosphate buffered saline with 0.05% Tween-20 (PBS-T20) and tapped dry.

(216) Serum was diluted 1/50 in buffer (Sigma #B6429) and 100 μl of this was added per well of the antigen coated plate, each sample tested in duplicate. The plate was incubated for 1 hr at room temperature on a rotary shaker at 160 rpm and then washed with PBS-T20 as described above.

(217) An HRP conjugated Protein-G conjugate (Thermo #31499), diluted to 1 μg/ml in buffer, was then added in 100 μl volumes to each well of the plate which was then incubated and washed as described above for serum. The plate was then developed with ABTS (2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt) and hydrogen peroxide substrate for 10-15 mins, stopped with 0.4 mM sodium azide and read at 405 nm wavelength. The optical density for the duplicates was averaged and the blank OD (buffer only instead of sera) was subtracted. This value was then expressed as a percentage of a common positive control serum sample from a B. abortus biovar 1 (A dominant) infected bovine for cattle sera.

(218) As expected, there was much cross reaction between the Brucella and Y. enterocolitica O:9 OPS antigens with both sets of sera, i.e., those from the cattle experimentally infected with Brucella abortus strain 544 and those experimentally infected with Y. enterocolitica O:9. An additional output was calculated for each sample by dividing the result from the B. melitensis OPS iELISA by that from the Y. enterocolitica O:9 OPS iELISA to derive a simple ratiometric value (henceforth described as the ‘Bm16M/YeO:9 Ratio’). Thus, higher Bm16M/YeO:9 Ratio values signify that the intensity of the reaction to the B. melitensis 16M OPS antigen was greater than the intensity of the reaction to the Y. enterocolitica O:9 antigen relative to lower Bm16M/YeO:9 Ratio values. Analysis of this data set revealed that there was a highly significant difference (P=0.008, Student's t-test for unpaired data) between the the Bm16M/YeO:9 Ratio values for the two different sample populations, whereby the mean value from the Brucella infected animals was higher than the mean value from the Y. enterocolitica O:9 infected animals. There was, therefore, a significant difference in the way that the serum from the two populations reacted to these two antigen types, with the sera from the Brucella infected animals reacting with a relatively higher intensity to the B. melitensis 16M OPS and vice versa. This result suggests that there are significant and detectable differences in the anti-OPS antibody repertoire between the two serum populations.

(219) As a consequence of this finding with the relative OPS ELISA results, the same methods were applied to archived bovine field sera from Great Britain. A total of 45 samples from individual animals confirmed by culture to be infected with B. abortus biovar 1 (A dominant) were tested. Also 68 samples were tested from individual animals whose sera was collected from 1996 to 1999, more than 10 years since the declaration of officially brucellosis free status for Great Britain, that were positive in conventional serology for brucellosis such as B. abortus S99 sLPS iELISA, SAT and CFT (Nielsen et al. (2009) “Bovine brucellosis” In: Manual of Diagnostic Tests & Vaccines for Terrestrial Animals 2009; Office International Des Epizooties, Paris, pg 10-19) but for which there was no cultural or epidemiological evidence of brucellosis. As with the data from the experimentally infected animals there was a highly significant difference (P=0.000012, Student's t-test for unpaired data) between the Bm16/YeO:9 Ratio values for the two different serological groups. As before, sera from cattle with confirmed brucellosis had, on average, higher values (FIG. 2).

(220) The results from both the B. melitensis and Y. enterocolitica O:9 OPS iELISAs were evaluated to find the positive/negative cut-off for each which generated the highest Youden Index (YI=diagnostic sensitivity [DSn]+diagnostic specificity [DSp]−1). These optimised YI values with the associate DSn and DSp figures are shown in Table 3.

(221) TABLE-US-00003 TABLE 3 Performance statistics for OPS iELISAs as tested on bovine sera Optimal Youden Index (YI = DSn + DSp − 1) ROC - Area Under Curve YI DSn DSp 95% Confidence Assay Estimate % % AUC Interval Y. enterocolitica 0.1297 64.44 48.53 0.5065 0.3964-0.6167 O:9 OPS iELISA B. melitensis 0.4232 55.56 86.76 0.7065 0.6024-0.8106 16M OPS iELISA Bm16M/YeO:9 0.5343 66.67 86.76 0.8056 0.7219-0.8892 Ratio

(222) To further evaluate the diagnostic effectiveness of each of the OPS iELISAs Receiver Operator Characteristic (ROC) Curve analysis was used, in particular the evaluation of the Area Under the Curve (AUC) (Hanley and McNeil (1982) Radiology 143, 29-36). In this context, the AUC represents the ability of the assay to correctly classify samples from animals that are Brucella infected and those that are not. The data in Table 2 shows that the B. melitensis OPS iELISA has a higher optimised YI and AUC value than the Y. enterocolitica O:9 OPS iELISA.

(223) In Table 4, P values relating to testing for significant differences between AUC data are presented. Testing for the significance of differences between AUC values was performed using the method for paired samples (Hanley and McNeil (1983) Radiology 148, 839-843).

(224) TABLE-US-00004 TABLE 4 Comparison of ROC AUC statistics for the OPS iELISAs as tested on bovine sera Assay Difference between AUC Y. enterocolitica B. melitensis 16M (P = [one-tailed]) O:9 OPS iELISA OPS iELISA B. melitensis 16M OPS iELISA <0.0001 Bm16M/YeO:9 <0.0001 0.239

(225) This data shows that there is a highly significant difference (P<0.0001) between the AUC values for the Y. enterocolitica O:9 and B. melitensis 16M OPS iELISAs and therefore the assay is significantly superior. The improvement in diagnostic performance when using the Bm16M/YeO:9 Ratio values is also demonstrated in Tables 3 and 4. The optimal YI value is greater than that for both of the individual OPS iELISA assays. The DSp is equal to that of the B. melitensis 16M OPS iELISA and the DSn is superior. The AUC is also greater, but not significantly so, compared to the AUC for the B. melitensis 16m OPS iELISA (P=0.239).

(226) This is strong evidence to demonstrate that a combinational ratiometric approach to the determination of infection status can be more advantageous than the interpretation of one test alone when there is significant cross reaction due to similar but non-identical antigens. The data for the B. melitensis 16M OPS iELISA alone and in combination as a ratiometric assay suggests that the M epitope is playing a major role in the significant differences in antibody binding that have been observed. The Bm16M/YeO:9 Ratio evaluation of the samples is a relatively crude attempt to delineate the contribution to overall titre made by the specific antibodies and epitopes from the contribution made by the common ones.

(227) The same OPS iELISA methods were also applied to 41 samples from individual swine that were positive to the Rose Bengal Test (RBT) and iELISA (Olsen, (2010) “Porcine Brucellosis” In: Office International Des Epizooties, Paris) and from herds confirmed by culture to be infected with B. suis biovar 1, an A dominant OPS biovar (Meikle et al. (1989) Infect Immun 57, 2820-2828; Olsen, (2009) “Porcine Brucellosis” In: Office International Des Epizooties, Paris, pages 3-4). A further 52 samples were tested which were collected from individual animals in Great Britain, officially free of B. suis, within herds which from which one or more sample positive in conventional serology such as RBT, cELISA, iELISA (Olsen, (2009) “Porcine Brucellosis” In: Office International Des Epizooties, Paris, pages 3-4) where obtained and where there was no epidemiological evidence of brucellosis.

(228) As with the data from the cattle sera samples, there was a highly significant difference (P=0.000000006, using the unpaired Student's t-test) between the Bm16/YeO:9 Ratio values for the two different serological groups. As before, sera from swine with confirmed brucellosis had a higher, on average, values than did the false positive serological samples (FIG. 3A). These samples were also tested by B. abortus S99 (A dominant) OPS iELISA (FIG. 3B) and a ratiometric expression of the data, analogous to the Bm16M/YeO:9 Ratio, was evaluated. There was a significant difference between the ratio of the B. abortus S99 to Y. enterocolitica O:9 OPS iELISA results for the different serum groups, but the difference was not as strong (P=0.023) as observed with the Bm16M/YeO:9 Ratio. In fact, the difference between the results from the two Brucella OPS antigens, B. melitensis 16M and B. abortus S99, was much more significant (P=0.000000016) than the difference between the Y. enterocolitica O:9 and B. abortus S99 results. This reflects the known structure of the these antigens with the A dominant OPS of B. abortus S99 having only approximately 2% α-1,3 linkages compared to the M dominant B. melitensis 16M OPS with 20% and Y. enterocolitica with 0%.

(229) The optimised YI, as described above, for the individual OPS iELISAs and the Bm16M/YeO:9 Ratio, results for the swine sera are shown in Table 5 where: Bm16M/YeO:9>B. melitensis 16M>B. abortus S99>Y. enterocolitica O:9.

(230) TABLE-US-00005 TABLE 5 Performance statistics for OPS iELISAs as tested on porcine sera Optimal Youden Index (YI = DSn + DSp − 1) ROC - Area Under Curve YI DSn DSp 95% Confidence Assay Estimate % % AUC Interval Y. enterocolitica 0.3588 87.80 48.08 0.6445 0.5328-0.7562 O:9 OPS iELISA B. abortus S99 0.6472 87.80 76.92 0.8607 0.7832-0.9382 OPS iELISA B. melitensis 0.7087 80.49 90.38 0.9135 0.8559-0.9710 16M OPS iELISA B. melitensis 0.7678 90.24 86.54 0.9085 0.8437-0.9733 16M/ Y. enterocolitica O:9

(231) The data for the AUC was similar whereby: B. melitensis 16M>Bm16MIYeO:9 Ratio>B. abortus S99>Y. enterocolitica O:9. The AUC for the Y. enterocolitica O:9 OPS iELISA was significantly lower (P<0.0001) than for the other three outputs (Table 6). There was a difference of weak significance between the AUC for the B. abortus and B. melitensis OPS iELISAs (P=0.066).

(232) TABLE-US-00006 TABLE 6 Comparison of ROC AUC statistics for the OPS iELISAs as tested on porcine sera Assay B. abortus B. melitensis Difference between AUC Y. enterocolitica S99 16M OPS (P = [one-tailed]) O:9 OPS iELISA OPS iELISA iELISA B. abortus S99 OPS <0.0001 iELISA B. melitensis 16M OPS <0.0001 0.066 iELISA Bm16M/YeO:9 <0.0001 0.174 0.452

(233) The same methods were applied to 21 serum samples from individual ELISA positive swine from herds confirmed by culture to be infected with B. suis biovar 2 and compared to the data from the 52 samples from non-infected swine, but seropositive herds, in Great Britain, as well as to the 41 samples from B. suis biovar 1 infected animals. There was no significant difference in the Bm16M/YeO:9 Ratio results between the sera from the B. suis biovar 2 infected animals and from sera from the non-infected animals (P=0.926). This is in keeping with the recent discovery that, uniquely for Brucella, the OPS from B. suis biovar 2 contains no α-1,3 linkages (Zaccheus et al. (2013) PLoS One 8, e53941) and therefore the OPS is highly similar, if not identical, to that of Y. enterocolitica O:9.

(234) The data from the swine sera demonstrates that assays using OPS with higher proportions of α-1,3 linkages provide superior diagnostic attributes. This is further evidence that the α-1,3 linkage is part of a significant and discriminating epitope for polyclonal sera derived from animals naturally infected with Brucella.

(235) Developing the Discrete M Epitope Antigen (Specific M-Antigen)

(236) The evidence described above from the OPS iELISAs provided the stimulus for additional studies to define, isolate and apply the ‘M’ epitope to the serodiagnosis of brucellosis in order to increase the accuracy of the results. The purified native OPS from Y. enterocolitica O:9, B. melitensis 16M (‘M’ dominant) and B. abortus S99 (‘A’ dominant) was partially hydrolysed using hot concentrated hydrochloric acid to obtain di- to dodeca-saccharides. These were evaluated by LC-ESI-MS/MS using a graphitized carbon column (Ruhaak et al. (2009) Anal. Bioanal. Chem. 394, 163-174) to separate them and confirm their identity as 4,6-dideoxy-4-formamido-α-D-mannopyranosyl oligomers.

(237) The oligosaccharides from the B. abortus S99 OPS were subjected to affinity chromatography using an affinity chromatography column with immobilised Brucella anti-M monoclonal antibody BM40 and the wash and elution fractions were further evaluated by LC-ESI-MS/MS using a graphitized carbon column (Ruhaak et al. (2009) Anal. Bioanal. Chem. 394, 163-174) to separate the oligosaccharides. Tetrasaccharides of 4,6-dideoxy-4-formamido-α-D-mannopyranosyl were readily detectable in the bound and subsequently eluted, using dilute hydrochloric acid, fractions; the LC-ESI-MS chromatogram was compared to those for the tetrasaccharides found in the non-affinity selected oligosaccharides derived from the partial acid hydrolysis of the three native antigens.

(238) Whereas the chromatogram for the B. melitensis 16M derived tetrasaccharide was relatively simple (FIG. 4A), those for B. abortus S99 (FIG. 4B) and Y. enterocolitica O:9 (FIG. 4C) were more complex. As the mass was the same across the chromatogram, the differences in elution profile were most likely due to changes in conformation and interaction with other oligosaccharides during separation within the graphitised column. What is evident is that the major peak found in the B. melitensis 16M tetrasaccharide chromatograph, eluting at about 9 mins, is also found in the B. abortus S99 but not the Y. enterocolitica O:9 chromatograph. The chromatograph of the B. abortus S99 tetrasaccharide affinity selected by the anti-M monoclonal antibody (FIG. 4D) looks extremely similar to the unselected tetrasaccharide from the acid hydrolysed native B. melitensis 16M OPS preparation. The conclusion from this study is that a tetrasaccharide from the Brucella OPS is large enough to form a viable antibody epitope and that the Brucella specific anti-M monoclonal antibody binds to a tetrasaccharide that is detectable in B. abortus and not in Y. enterocolitica O:9. From the pre-existing structural knowledge, the only known difference is the α-1,3 linkage.

(239) All the evidence presented above provided a good basis for the hypothesis that a tetrasaccharide antigen containing the M epitope, whilst minimising any C/Y or A epitope-like properties, would make a useful serodiagnostic antigen. Four 4,6-dideoxy-4-formamido-α-D-mannopyranose residues were synthesised (Sussex Research Laboratories Inc, Ottawa, Ontario, Canada) within a tetrasaccharide that is sequentially α-1,2, α-1,3 and α-1,2 linked, generating a tetrasaccharide having Formula VIII:
4,6-dideoxy-4-formamido-α-D-mannopyranosyl-(1-2)-4,6-dideoxy-4-formamido-α-D-mannopyranosyl-(1-3)-4,6-dideoxy-4-formamido-α-D-mannopyranosyl-(1-2)-4,6-dideoxy-4-formamido-α-D-mannopyranose   (Formula VIII)
This molecule had not previously been synthesised and applied and is referred to below as the TSM antigen (the “tetrasaccharide M [like] antigen”). The key aspect of this structure is the inclusion of the α-1,3 linkage whilst incorporating the minimal number of α-1,2 linkages thought necessary for M specific antibodies to bind; this has the effect of minimising the binding of non-M specific antibodies.
Analysis of the TSM Antigen

(240) The structure of the antigen was confirmed by MALDI-ToF and ESI-QToF mass spectrometry. The monoclonal binding properties were confirmed by competitive ELISA whereby a standard polystyrene ELISA plate was coated with B. melitensis 16M sLPS antigen by passive absorption in carbonate buffer. The plate was co-incubated with the anti-M monoclonal antibody BM40 and native OPS from B. melitensis 16M, B. abortus S99, Y. enterocolitica O:9, the TSM antigen as well as buffer controls. After washing the plate, an HRP conjugate specific for the BM40 was added and incubated. After washing, the plates were developed with an HRP substrate to determine the degree to which mAb-conjugate binding had been inhibited.

(241) The results (FIG. 5) demonstrated that the TSM antigen was able to inhibit the binding to a greater extent than the equivalent concentration of Y. enterocolitica O:9 OPS and to a similar degree as the B. abortus S99 OPS. Taking into account the monovalency of the TSM antigen compared to the multiple binding sites available on 16M OPS, the results provide strong evidence for specific TSM-antibody binding.

(242) A similar competitive ELISA was conducted to measure the inhibition of binding of anti-A and anti-M rabbit hyperimmunised and absorbed monospecific sera (sera as described previously) using native OPS from B. melitensis 16M, B. abortus S99, Y. enterocolitica O:9, the TSM antigen as well as buffer controls. The results demonstrated preferential inhibition of the anti-M sera by the TSM (FIG. 6A) compared to the inhibition of the anti-A sera (FIG. 6B), in agreement with the theoretical expectations.

(243) Application of the TSM Antigen to Serodiagnosis

(244) The TSM antigen was then chemically conjugated to an ELISA plate to create an indirect ELISA and enable the evaluation of non-competitive antibody interactions. Owing to the highly soluble nature of the antigen and its small size, passive absorption to an ELISA plate surface was not straightforward. Instead, it was chemically conjugated to an ELISA plate surface functionalised with hydrazide groups on the end of spacer arms (Corning Carbo-BIND™ #2507). As described above, the hydrazide groups react spontaneously with aldehydes that may be created within carbohydrates by periodate oxidation.

(245) The TSM antigen was incubated in 2 mM sodium metaperiodate, 50 mM sodium acetate buffer at pH 5.5 for 2.5 hours in the dark at 4° C. This was sufficient to oxidise the vicinal diol hydroxyl groups on the 2.sup.nd and 3.sup.rd carbons of the terminal sugar (Sussich et al. (2000) Carbohydr Res 329, 87-95), the only vicinal diol group within the molecule, to generate the structure shown below:

(246) ##STR00025##

(247) Competitive ELISA with BM40 anti-M monoclonal antibody was applied as described above to confirm that the oxidised TSM antigen (oxiTSM) shown above maintained its antibody interaction. Residual sodium metaperiodate was removed from the reaction by filtration of the TSM antigen and buffer through a short column of Sephadex G-10 (GE Healthcare) which reacted with the excess sodium metaperiodate and, with a suitable volume of elution buffer, allowed the oxiTSM antigen to flow through. Removal of the sodium metaperiodate was necessary to prevent any further oxidation of the oxiTSM and removal was confirmed by abrogation of the reaction with ABTS (2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt).

(248) The oxiTSM antigen was then diluted to 10 μg/ml in 0.1 M sodium acetate buffer pH 5.5 and 100 μl was added per well to a Carbo-BIND™ plate which was incubated at 37° C. for 3 hrs. During this incubation process the aldehyde groups on the oxidised tetrasaccharide spontaneously reacted with the hydrazide groups on the end of a linker attached to the ELISA plate surface to form stable covalent hydrazone bonds, as shown below (the hydrazone bond is shown with a dotted circle):

(249) ##STR00026##

(250) After this time the plate was washed with 4 times 200 μl per well of phosphate buffered saline with 0.5% Tween-20 (PBS-T20) and tapped dry. These coated ELISA plates were used to test the field sera from cattle (described above) confirmed by culture to be infected with B. abortus (n=45) and from cattle without brucellosis but with serum cross reacting in one or more conventional serodiagnostic assay (n=68).

(251) Serum was diluted 1/50 in buffer (Sigma #B6429) and 100 μl of this was added per well of the antigen coated plate, each sample tested in duplicate. The plate was incubated for 1 hr at room temperature on a rotary shaker at 160 rpm and then washed with PBS-T20 as described above. An HRP conjugated Protein-G conjugate (Thermo #31499), diluted to 1 μg/ml in buffer, was then added in 100 μl volumes to each well of the plate which is then incubated and washed as described above for serum. The plate was then developed with ABTS (2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt) and hydrogen peroxide substrate for 10-15 mins, stopped with 0.4 mM sodium azide and read at 405 nm wavelength. The optical density for the duplicates was averaged and the blank OD (buffer only instead of sera) was subtracted. This value was then expressed as a percentage of a common positive control serum sample from a Brucella infected bovine.

(252) The TSM antigen iELISA was evaluated against the same population of bovine samples as described above for the evaluation of the B. melitensis 16M and Y. enterocolitica O:9 OPS iELISAs: 45 from B. abortus biovar 1 (A dominant) culture positive animals and 68 from false positive serological reactors. The results for the ELISA are shown in FIG. 7.

(253) Further Antigen Oligosaccharide Conjugates—ELISA Titrations

(254) To begin to determine the overall length of the oligosaccharide necessary to provide specificity for binding to anti-A or anti-M antibodies, synthetic pentasaccharide and nonasaccharide BSA conjugate antigens were evaluated against the bovine sera described above. Each antigen contains one internally and centrally positioned α-1,3 link with the remaining links being α-1,2.
The pentasaccharide had Formula XI:
4,6-dideoxy-4-formamido-α-D-mannopyranosyl-(1-2)-4,6-dideoxy-4-formamido-t-D-mannopyranosyl-(1-3)-4,6-dideoxy-4-formamido-α-D-mannopyranosyl-(1-2)-4,6-dideoxy-4-formamido-α-D-mannopyranosyl-(1-2)-4,6-dideoxy-4-formamido-α-D-mannopyranose   (Formula XI)
The nonasaccharide had Formula XV:
4,6-dideoxy-4-formamido-α-D-mannopyranosyl-(1-2)-4,6-dideoxy-4-formamido-α-D-mannopyranosyl-(1-2)-4,6-dideoxy-4-formamido-α-D-mannopyranosyl-(1-2)-4,6-dideoxy-4-formamido-α-D-mannopyranosyl-(1-3)-4,6-dideoxy-4-formamido-D-mannopyranosyl-(1-2)-4,6-dideoxy-4-formamido-α-D-mannopyranosyl-(1-2)-4,6-dideoxy-4-formamido-α-D-mannopyranosyl-(1-2)-4,6-dideoxy-4-formamido-α-D-mannopyranosyl-(1-2)-4,6-dideoxy-4-formamido-α-D-mannopyranose   (Formula XV)
In Formulae XI and XV above, the central 4,6-dideoxy-4-formamido-D-mannopyranose is underlined. The antigens were conjugated to BSA via a reducing end -1-O—(CH.sub.2).sub.5—COO—CH.sub.3 linker as described above, to form structures 42 and 43, respectively, shown below.

(255) ##STR00027##

(256) In one experiment, BSA-carbohydrate-protein conjugates 42 and 43 (5 g/mL in PBS) were used to coat 96-well microtiter plates (MaxiSorp, Nunc) overnight at 4° C. The plate was washed 5 times with PBST (PBS containing 0.05% (v/v) Tween 20). Serial √10 dilutions of mAb YsT9.1 and Bm10 ascites fluids or supernatants form hybridoma cell culture were made in PBST containing 0.1% BSA. The solutions were distributed in duplicate on the coated microtiter plate and incubated at room temperature for 2 hours. The plate was washed with PBST (5 times) and goat anti-mouse IgG antibody conjugated to horseradish peroxidase (Kirkegaard & Perry Laboratories; 1:2000 dilution in 0.1% BSA/PBST; 100 L/well) was added. The mixture was then incubated for 1 hour. The plate was washed 5 times with PBST before addition of a 1:1 mixture 3,3′,5,5′-tetramethylbenzidine (0.4 g/L) and 0.02% H.sub.2O.sub.2 solution (Kirkegaard & Perry Laboratories; 100 L/well). After 2 minutes, the reaction was stopped by addition of 1 M phosphoric acid (100 L/well). Absorbance was read at 450 nm. End point titres are recorded as the dilution giving an absorbance 0.2 above background.

(257) The monoclonal antibodies (YsT9-1 and BM10) previously shown to be specific for the Brucella A and M antigens (Bundle et al. (1989) Infect. Immun. 57, 2829-2836). were titred to their end point against the antigens 42 and 43 coated on ELISA plates (FIG. 8). The nonasaccharide antigen 43 binds anti-A and M specific antibodies with equivalent avidity, whereas the pentasaccharide 42 displays a preference for the M specific antibody, while still binding the A specific antibody but with an approximately 10 fold reduced avidity.

(258) Previous studies showed the YsT9-1 and Bm10 antibodies possessed avidity differences of between 400-1,000 for the respective O-polysaccharide antigens (Bundle et al. (1989). Infect. Immun. 57, 2829-2836). As mentioned, the pentasaccharide antigen 42 shows a preference for M specific antibody.

(259) Further Antigen-Oligosaccharide Coniugates—Serology Studies and Analysis

(260) In a further experiment, the conjugates 42 and 43 were immobilised onto the surface of standard polystyrene ELISA plates passively via overnight incubation in carbonate buffer at 4° C. at 2.5 g/ml, 100 μl/well. The plates were washed as described above and incubated with a 1/50 dilution of sera in buffer (in duplicate)(see below) for 30 mins at room temperature at 160 rpm, after which time they were washed and tapped dry as described above. For bovine sera, an HRP-conjugated mouse anti-bovine IgG conjugate was used. The conjugates were diluted to working strength in buffer and the plates incubated, washed and tapped dry as for the serum incubation stage. The plate was then developed with ABTS (2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt) and hydrogen peroxide substrate for 10-15 mins, stopped with 0.4 mM sodium azide and read at 405 nm wavelength. The optical density for the duplicates was averaged and the blank OD (buffer only instead of sera) was subtracted. This value was then expressed as a percentage of a common positive control serum sample from a Brucella infected bovine.

(261) The same method employed to evaluate bovine serum by iELISA with the pentasaccharide and nonasaccharide conjugates was used to evaluate bovine serum by a further four oligosaccharide BSA conjugates. The oligosaccharides were:
4,6-dideoxy-4-formamido-α-D-mannopyranosyl-(1-2)-4,6-dideoxy-4-formamido-α-D-mannopyranosyl-(1-3)-4,6-dideoxy-4-formamido-α-D-mannopyranosyl-(1-2)-4,6-dideoxy-4-formamido-α-D-mannopyranose   (tetrasaccharide, Formula VIII)
4,6-dideoxy-4-formamido-α-D-mannopyranosyl-(1-3)-4,6-dideoxy-4-formamido-α-D-mannopyranosyl-(1-2)-4,6-dideoxy-4-formamido-α-D-mannopyranosyl   (trisaccharide (terminal α-1,3 link), Formula XVI)
4,6-dideoxy-4-formamido-α-D-mannopyranosyl-(1-2)-4,6-dideoxy-4-formamido-α-D-mannopyranosyl-(1-3)-4,6-dideoxy-4-formamido-α-D-mannopyranosyl   (trisaccharide (terminal α-1,2 link), Formula XVII)
4,6-dideoxy-4-formamido-α-D-mannopyranosyl-(1-3)-4,6-dideoxy-4-formamido-α-D-mannopyranose   (disaccharide, Formula II)
These antigens were conjugated to BSA via a reducing end -1-O—(CH.sub.2).sub.5—COO—CH.sub.3 linker as described above, to form structures similar to 42 and 43 shown above. Since conjugation occurs via the reducing end, the link at the non-reducing end is referred to as the “terminal link”.

(262) The BSA-nonasaccharide, pentasaccharide, tetrasaccharide, trisaccharide and disaccharide conjugate iELISAs (and the oxiTSM iELISA described above) were evaluated against the same population of bovine field samples as described above for the evaluation of the B. melitensis 16M and Y. enterocolitica O:9 OPS iELISAs; that is, 45 from B. abortus biovar 1 (A dominant) culture positive animals and 68 from false positive serological reactors. The results for these iELISAs, shown by scatter plot in FIGS. 7 and 9 to 11, were evaluated to find the optimal Youden Index for each iELISA and ROC analysis was used to determine the ROC Curves and AUC. This data is presented in Table 7 below, along with the 95% confidence intervals for the AUC; Table 7 shows the data from Table 3 and the additional oligosaccharide data, for ease of comparison. Table 8 shows the probability (P) values for the testing of statistically significant differences between the AUC data from each antigen assay. The AUCs were all compared against each other and the P values was calculated using the Normal Distribution model. The evaluation was 1-tailed as the prior hypothesis under investigation was that antigens that were more ‘M’ like would have higher AUC values. As with Table 7, Table 8 shows the earlier information from Table 4 with the additional oligosaccharide data.

(263) A selection of ROC curves are shown in FIG. 12. The Figure shows the ROC curves for both native antigens tested and amply demonstrates the superiority of the B. melitensis 16M OPS over the Y. enterocolitica O:9 OPS. The data from the M-like synthetic BSA-pentasaccharide, BSA-tetrasaccharide and BSA-disaccharide conjugate iELISAs is also shown and graphically depicts the improvement in diagnostic performance that is commensurate with the reduction of α-1,2 linkages in the structures and retention of the α-1,3 link.

(264) TABLE-US-00007 TABLE 7 Performance statistics for OPS and Synthetic Oligosaccharide iELISAs as tested on bovine sera Optimal Youden Index % (YI = DSn + DSp − 1) ROC - Area Under Curve age YI 95% α-1,3 Esti- DSn DSp Confidence Assay links mate % % AUC Interval Y. entero- 0 0.1297 64.44 48.53 0.5065 0.3964-0.6167 colitica O:9 OPS iELISA B. melitensis 20 0.4232 55.56 86.76 0.7065 0.6024-0.8106 16M OPS iELISA Nona- 12.5 0.5713 68.89 88.24 0.8317 0.7512-0.9122 saccharide iELISA Penta- 25 0.6174 95.56 66.18 0.8667 0.8019-0.9314 saccharide iELISA Tri- 50 0.6091 73.33 86.76 0.8672 0.7976-0.9367 saccharide (terminal α-1,2) iELISA oxiTSM 33.3/50 0.6670 73.33 92.65 0.8948 0.8345-0.9550 iELISA Tri- 50 0.7268 80.00 92.65 0.8987 0.8334-0.9640 saccharide (terminal α-1,3) iELISA Tetra- 33.3 0.6745 73.33 94.12 0.9046 0.8478-0.9613 saccharide iELISA Di- 100 0.7860 84.44 92.65 0.9310 0.8345-0.9550 saccharide iELISA

(265) TABLE-US-00008 TABLE 8 Comparison of ROC AUC statistics for the OPS and Synthetic Oligosaccharide iELISAs as tested on bovine sera Difference between AUC (P = [one-tailed]) YeO9 Bm Bm/Ye NS PS Tri t1,2 OxiTSM Tri t1,3 Tetra Y. enterocolitica O:9 OPS iELISA (YeO9) B. melitensis 16M OPS iELISA <0.0001 (Bm) Bm16M/YeO:9 (Bm/Ye) <0.0001 0.0239 Nonasaccharide iELISA (NS) <0.0001 0.0032 0.3050 Pentasaccharide iELISA (PS) <0.0001 0.0005 0.0918 0.1003 Trisaccharide (terminal α-1,2) <0.0001 0.0007 0.1093 0.2148 0.4920 iELISA (Tri t1,2) oxiTSM iELISA (oxiTSM) <0.0001 <0.0001 0.0239 0.0307 0.1867 0.2005 Trisaccharide <0.0001 <0.0001 0.8080 0.0384 0.1736 0.1949 0.4522 (terminal α-1,3) iELISA (Tri t1,3) Tetrasaccharide iELISA (Tetra) <0.0001 <0.0001 0.0228 0.0146 0.0853 0.0359 0.3632 0.4207 Disaccharide iELISA (Di) <0.0001 <0.0001 0.0158 0.0087 0.0322 0.0322 0.1379 0.0901 0.2005

(266) The data presented in Table 7 show the antigens in ascending AUC value (lowest at the top and highest at the bottom). In this context, the AUC represents the ability of the assay to correctly classify samples from animals that are Brucella infected and those that are not. In this data analysis, all the samples from animals that were not Brucella infected were falsely positive in one or more conventional serodiagnostic assays for brucellosis. The results (based on the AUC data) show that the B. melitensis OPS is superior to the Y. enterocolitica O:9 OPS and it is proposed that this is due to the presence of α-1,3 links within the OPS of the former. The percentage of links within the antigenic structures that are α-1,3 (the remainder being α-1,2) is also shown in the table.

(267) In general, the AUC values increase with the increase in the percentage of α-1,3 links within the antigen (and decrease in α-1,2 links). This is clearly evident in the comparison of the two native OPS antigens and is also evident in the AUC data for the nonasaccharide, pentasaccharide, tetrasaccharide and disaccharide BSA conjugates. There are, however, some nuances within the data that should be considered as described below with regard to the comparison between the nonasaccharide BSA conjugate and B. melitensis 16M OPS, the oxidised tetrasaccharide antigen and the two trisaccharide BSA conjugates.

(268) All the synthetic BSA conjugated oligosaccharide antigens have superior diagnostic capability in this regard compared to the native OPS antigens. This includes the nonasaccharide BSA-conjugate which has proportionally fewer α-1,3 links than the B. melitensis 16M OPS. This apparent anomaly may due to do with the precise positioning of the α-1,3 links within the native structure and the multivalent nature in which antibodies may bind this structure.

(269) It is not straightforward to evaluate the performance of the oxiTSM antigen relative to the others investigated due to the methodological differences, not least the breaking of the terminal perosamine, linking directly to a functionalised ELISA plate surface from the remnants of this structure and thus presenting the reducing sugar as the tip of the antigen. For this reason, the percentage of α-1,3 links presented in Table 7 is shown as 33.3/50 dependent upon the undetermined significance of the link to the oxidised terminal “perosamine”. Despite these apparent impediments, the oxiTSM iELISA possessed a greater AUC value than the BSA-pentasaccharide and α-1,2 terminated BSA-trisaccharide conjugate iELISAs. This may be due to the loss of the terminal end ‘tip’ epitope, since this is not presented by the oxiTSM antigen due to the oxidation and conjugation of the terminal sugar. The natural ‘tip’ antigen would be similar in the OPS from A and M dominant Brucella and Y. enterocolitica O:9. This ‘tip’ epitope may also explain the higher AUC value for the α-1,3 terminated trisaccharide compared to the α-1,2 terminated trisaccharide. According to the structural scheme recently presented (Kubler-Kielb & Vinogradov (2013) Carbohydr. Res. 378, 144-147), the tip of most OPS molecules in M dominant OPS is a α-1,2 linked disaccharide, as would also be the case in the OPS from A dominant Brucella strains and from Y. enterocolitica O:9. The α-1,3 terminated trisaccharide does not present such a tip and thus common anti-‘tip’ epitope antibodies may be less likely to bind in comparison to the antibodies against the linear M epitope.

(270) The highest AUC value is generated by the BSA-disaccharide conjugate iELISA. This was significantly higher (P=0.0322) than the AUC value for the BSA-pentasaccharide conjugate iELISA which was itself significantly higher than the AUC value derived from the native antigens (Y. enterocolitica O:9 and B. melitensis 16M OPS) The disaccharide has no α-1,2 links present, just a single α-1,3 link. On the basis of this data, this structure is highly functional and represents the minimal size M epitope. The ability of such a small structure to bind to so many polyclonal antibodies and to do so in such a selective manner is a surprising finding. The negative impact of even a single α-1,2 link was just as unexpected. The ability and the extent to which the disaccharide (and the other M-like oligosaccharides), can selectively bind to polyclonal sera raised by infection with A dominant strains of Brucella was also unexpected, but has been demonstrated here for the first time.

(271) The data from the Max YI values largely agree with the AUC data in that the smaller BSA-oligosaccharides conjugates with fewer α-1,2 links provide superior diagnostic parameters. The main difference is the superior Max YI value for the α-1,3 terminated BSA-trisaccharide conjugate compared to the BSA-tetrasaccharide conjugate. This can be rationalised by the arguments put forward above with regards to the reduction of the ‘tip’ epitope as well as the elimination of a α-1,2 link.

(272) All of the BSA-oligosaccharide conjugate iELISAs were also applied to the sera from the eight animals, described above, that were experimentally infected with either B. abortus strain 544 (n=4) or Y. enterocolitica O:9 (n=4). Only samples collected from weeks 3, 7, 16 and 24 were tested because the samples collected from the B. abortus infected animals at week 53 gave ambivalent results with conventional serodiagnostic assay.

(273) The results for the samples from the experimentally infected cattle are shown in FIGS. 13 to 19. FIG. 13 shows the results from B. melitensis 16M OPS iELISA and demonstrates that there is a considerable response from the sera derived from the Y. enterocolitica O:9 infected animals. FIGS. 14 and 15 show the data derived from the nonasaccharide and pentasaccharide BSA conjugate iELISAs respectively. The results from the B. melitensis 16M OPS iELISA and pentasaccharide BSA conjugate iELISA are shown against each other in a simple scatter plot in FIG. 16. FIGS. 17 and 18 show the data derived from the tetrasaccharide and disaccharide BSA conjugate iELISAs respectively. Although the ability to differentiate between the antibodies derived from the two infection types is not absolute, FIG. 18 shows that the disaccharide is close to achieving this aim. This differentiation is more readily visible in the scatter plot, FIG. 19, showing the results from the tetrasaccharide and disaccharide BSA conjugate iELISAs.

(274) All the serological data from the samples from the experimentally infected animals (weeks 3, 7, 16 and 24) were evaluated by the following quantitative criteria. The percentage (of 16) samples from the cattle experimentally infected with Y. enterocolitica O:9 with quantitatively greater serological titres than the lowest titre sample from cattle experimentally infected with B. abortus strain 544 was calculated. The percentage is shown, for each serodiagnostic assay, in Table 9. The CFT, SAT, sLPS iELISA and cELISA and FPA data has been published previously (McGiven et al. (2008) J. Immun. Meth. 20, 7-15) and shows the significant degree of cross reaction that occurs with conventional and contemporary serology.

(275) Although the data set is relatively small, the results show that the disaccharide and the trisaccharide BSA conjugate iELISAs (using the trisaccharide with a α-1,3 link at the non-conjugated terminus) were the best at differentiating between antibodies derived from the two types of infection. This demonstrates that as well as providing improved diagnostic specificity when testing field FPSRs, the disaccharide has the same beneficial effect with the samples that have been experimentally infected with a Gram-negative bacteria in possession of the OPS structure that is most similar to that of Brucella. In this sample set, the results from the nonasaccharide, pentasaccharide, tetrasaccharide and α-1,2 terminated trisaccharide BSA conjugate iELISAs showed no advantage over the B. melitensis 16M OPS iELISA. The two worst performing assays in this respect, both with a percentage of 81.25, were the iELISAs performed using the sLPS from B. abortus S99 (an A dominant strain) and the Y. enterocolitica O:9 OPS iELISA. This reflects the high degree of similarity between the OPS structures of these organisms.

(276) TABLE-US-00009 TABLE 9 Percentage (of 16) samples from cattle experimentally infected with Y. enterocolitica O:9 with quantitatively greater serological titres than the lowest titre sample from cattle experimentally infected with B. abortus strain 544 Percentage of samples Classical tests and native antigens Complement Fixation Test 31.25 Serum Agglutination Tests 56.25 iELISA (sLPS, B. abortus S99) 81.25 cELISA (sLPS, B. melitensis 16M) 43.75 Fluorescence Polarisation Assay 50.00 Y. enterocolitica O:9 OPS iELISA 81.25 B. melitensis 16M OPS iELISA 56.25 Synthetic oligosaccharide antigens Nonasaccharide 56.25 Pentasaccharide 75.00 Tetrasaccharide 75.00 Trisaccharide (terminal 1, 2) 68.75 Trisaccharide (terminal 1, 3) 18.75 Disaccharide 12.50

(277) The nonasaccharide, pentasaccharide, tetrasaccharide and disaccharide BSA-conjugate iELISAs were also evaluated against 125 serum samples from 125 randomly sampled non-Brucella infected cattle. The results are presented by scatter plot, with the results from the Brucella infected cattle (n=45), in FIGS. 20 (nonasaccharide and pentasaccharide) and 21 (tetrasaccharide and disaccharide).

(278) TABLE-US-00010 TABLE 10 Performance statistics for Synthetic Oligosaccharide iELISAs as tested on bovine sera(randomly sampled non-Brucella infected) Optimal Youden Index (YI = DSn + ROC - Area DSp − 1) Under Curve YI 95% Esti- Confidence Assay mate DSn % DSp % AUC Interval Nonasaccharide 0.9920 100.0 99.20 0.9998 0.9992-1.000  iELISA Pentasaccharide 1.0000 100.0 100.0 1.0000 1.000-1.000 iELISA Tetrasaccharide 0.9476 95.56 99.20 0.9964 0.9911-1.000  iELISA Disaccharide 0.8142 82.22 99.20 0.9108 0.8401-0.9814 iELISA

(279) The results, shown in scatter plot FIGS. 20 and 21 and Table 10, demonstrate that the nonasaccharide, pentasaccharide and tetrasaccharide BSA-conjugate iELISAs are highly effective serodiagnostic assays. The data also suggests that the disaccharide BSA-conjugate iELISA is less effective in differentiating between these sample types than the other, larger, oligosaccharides in possession of α-1,2 links. When the disaccharide BSA-conjugate is used, the sera from the randomly sampled non-Brucella infected cattle have a much more similar response to that observed for the FPSR samples than is the case with the nonasaccharide, pentasaccharide and tetrasaccharide BSA-conjugate iELISAs.

(280) The oxiTSM, BSA-pentasaccharide and BSA-nonasaccharide antigens have also been applied to the detection of specific anti-Brucella antibodies in small ruminant sera. The oxiTSM antigen was conjugated to the Carbo-BIND™ ELISA plates by the same method as described above and the BSA conjugated oligosaccharides were coated to ELISA plates by the same method as described above. The assay was completed using the same method as described for bovine sera above except for the use of a protein G HRP conjugate. In total, 61 samples were evaluated from individual sheep and goats from flocks confirmed as infected with B. melitensis biovar 3 (mixed ‘A’ and ‘M’ dominance) and positive in iELISA using B. melitensis sLPS antigen. Also tested were 94 sera from sheep and goats from Great Britain, that has always been free of B. melitensis (FIG. 22).

(281) In FIG. 22, the lowest x-axis value for a sample from the Brucella infected population is 32.5, so there is no data from this population hidden in the overplotting of the data from the non-Brucella infected population. The optimised YI value for the 16M OPS iELISA was 0.984 (95% CI=0.952−1.000) and that of the ‘M’ tetrasaccharide iELISA was 0.816 (95% CI=0.720−0.912). The 95% confidence intervals for two YI values do not overlap, demonstrating a significant difference in diagnostic performance. However, the data does demonstrate that the TSM antigen does detect anti-Brucella antibodies within sera from small ruminants infected with B. melitensis. The data in FIG. 23 demonstrates the effectiveness of the universal nonasaccharide conjugate antigen as, based on this sample set, the DSn and DSp were both 100%.

(282) Finally, ELISA plates were coated with co-povidone disaccharide conjugates 99a and 99b as described above for BSA conjugates. Serial √10 dilutions of human sera from a patient infected with Brucella suis was applied to the plate and bound antibody was detected by a goat anti human horse radish peroxidase conjugate. The results are shown in FIG. 24. This demonstrated that the disaccharide can detect infection by Brucella in a sample from a human patient.

(283) Antibody Binding Studies

(284) BSA conjugates 98 (comprising the oligosaccharide of Formula XIV) and copovidone conjugate 100b (comprising an oligosaccharide which is exclusively α-1,2 linked) were each coated on ELISA plates as described above. Serial √10 dilutions of mouse monoclonal antibodies that are A or M specific were applied to the plate and bound antibody was detected by a goat anti mouse IgG horse radish peroxidase conjugate. Antibodies YsT9.1 and YsT9.2 are anti-A antibodies. Antibodies BM10 and BM28 are M-specific. The results are shown in FIGS. 25 and 26 and demonstrate the binding specificity of the disaccharide, as well as the failure of an exclusively α-1,2 linked oligosaccharide conjugate to bind to an anti-M antibody.

(285) To evaluate the binding of an anti-M specific mAb to the BSA-tetrasaccharide, trisaccharide and disaccharide conjugates, they were each coated to ELISA plates at 5 pg per ml (in pH 10.0 carbonate buffer), double diluted in neighbouring ELISA plate wells and the plates were incubated and washed as described previously for the bovine serum iELISAs with the BSA conjugates. A working strength dilution of HRP-conjugated BM40 mAb was added to each well and this was incubated for 30 mins at room temperature and then washed as described previously. The plates were developed with HRP ABTS substrate and the reaction stopped after 15 mins by addition of sodium azide. The ELISA plates were read at 405 nm to measure the optical density of each well.

(286) The results (FIG. 27) show that the BM40 mAb binds to the BSA conjugates; most effectively to the trisaccharide and also, almost equally, to the tetrasaccharide and disaccharide BSA conjugates. The binding of the BM40 mAb to these three BSA-oligosaccharide structures demonstrates the presence of the M epitope within them as BM40 has been previously shown to be highly specific to M dominant OPS. As well as binding BM40 mAb, the inventors have shown that these structures also bind polyclonal sera. Therefore, it would be possible to simultaneously incubate both the HRP conjugated BM40 mAb and serum antibodies in order to create a competitive ELISA for the detection of polyclonal antibodies in serum. The ability to do this with the disaccharide is particularly noteworthy as this is already the antigen that provides the best resolution of FPSRs, incorporating a competitive element into the immunoassay is expected to yield further improvements in diagnostic capability.

(287) Vaccination Study

(288) A vaccine glycoconjugate comprising an oligosaccharide formed by exclusively -(1-2)- linked 4,6-dideoxy-4-acylamido-α-pyranose units, the oligosaccharide covalently linked to a carrier protein such as tetanus toxin, is evaluated for vaccine efficacy as follows. The procedures are based upon the standard model of vaccine efficacy testing as described with the OIE Manual of Diagnostic Tests and Vaccines (Nielsen et al. (2009) In: Manual of Diagnostic Tests & Vaccines for Terrestrial Animals 2009; Office International Des Epizooties, Paris, Chapter: Bovine brucellosis, pg 22-29). Control and challenge groups of mice are established for evaluation. For example, control group 1—‘vaccination’ with PBS only; control group 2—vaccination with unconjugated ‘A’ oligosaccharide and tetanus toxoid plus adjuvant; control group 3—subcutaneous vaccination with 1×10.sup.5 CFU per mouse of reference vaccine B. abortus strain S19. The vaccine challenge groups are group 4—vaccination with type ‘A’ oligosaccharide tetanus toxoid glycoconjugate; and group 5—vaccination with type ‘M’ oligosaccharide tetanus toxoid glycoconjugate. Following vaccination mice are challenged by intraperitoneal inoculation with 2×10.sup.5 CFU (per mouse) of B. abortus strain 544. At 15 days post challenge mice are euthanised and the number of Brucella cells within their spleens enumerated to provide a metric of protection. The mice are also bled and serology performed to determine the antibody response from each group to the ‘A’ and ‘M’ epitopes. This provides a means by which to evaluate the DIVA potential of the oligosaccharide tetanus toxoid glycoconjugate.