Synthesis of diverse glycosylphosphatidylinositol glycans from Toxoplasma gondii and their application as vaccines and diagnostics

Abstract

The present invention relates to the synthesis of GPI-related surface antigens of the parasite Toxoplasma gondii (T. gondii) and the resulting products obtained. These synthetic compounds are suitable for diagnosis of toxoplasmosis, as well as vaccine against toxoplasmosis, a diseases caused by infection with T. gondii.

Claims

1. A compound of a formula (I): ##STR00021## wherein R represents ##STR00022## R.sup.1 is —OH and R.sup.4 is —OP(O)(OH)—O—X—NH.sub.2; X is selected from the group consisting of —CH.sub.2—, —C.sub.2H.sub.4—, —C.sub.3H.sub.6—, —C.sub.4H.sub.8—, —C.sub.5H.sub.10—, and —C.sub.6H.sub.12—; R.sup.2 represents —OP(O)(OR.sup.5)(OR.sup.6); R.sup.3 is selected from the group consisting of —H, —OH, —NH.sub.2, NHCOCH.sub.3, —NHCOCH.sub.2CH.sub.3, —NHCOCH.sub.2CH.sub.2CH.sub.3, and —N.sub.3; R.sup.5 and R.sup.6 are independently selected from the group consisting of —H, -L-SH, —(C.sub.2H.sub.4O).sub.r—CH.sub.2—SH and —(C.sub.2H.sub.4O).sub.r—C.sub.2H.sub.4—SH, with the proviso that R.sup.5 and R.sup.6 cannot both be —H, and one of R.sup.5 and R.sup.6 is hydrogen; L is a linking group; and r is an integer of from 1 to 40.

2. The compound of claim 1, wherein R.sup.5 is —H and R.sup.6 is —C.sub.6H.sub.12—SH.

3. The compound of claim 1, covalently linked to a carrier.

4. The compound of claim 3, wherein the carrier is selected from the group consisting of a diphtheria toxoid, a mutated diphtheria toxoid, a modified diphtheria toxoid, and a tetanus toxoid.

5. The compound of claim 3, immobilized on a carrier material by covalent bonding.

6. The compound of claim 5, wherein the carrier material is selected from the group consisting of a glass slide, a microtiter plate, test tubes, microspheres, nanoparticles and beads.

7. A method of vaccination, comprising: administering the compound of claim 3 to a patient, whereby the patient is vaccinated against toxoplasmosis.

8. A method of vaccination, comprising: administering the compound of claim 4 to a patient, whereby the patient is vaccinated against toxoplasmosis.

9. A method of vaccination, comprising: Administering the compound of claim 2 covalently linked to a carrier to a patient, whereby the patient is vaccinated against toxoplasmosis.

10. The compound of claim 1, having a formula: ##STR00023##

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1: Reaction scheme for the preparation of glycans 3, 4 and 5 as examples for a phosphate linked GPI.

(2) FIG. 2: Reaction scheme for the preparation of glycan 22 as example for a sulfone linked GPI.

(3) FIG. 3: left side: Analysis of pooled serum that was obtained 4 weeks after 1.sup.st immunization of Balb/c mice with a conjugate consisting of CRM.sub.197 covalently linked to compound 3; right side: Printing pattern: all compounds were printed in a 3×3 pattern and in a concentration of 1 mM; left, upper corner: compound 5; left lower corner: compound 3; right, upper corner: compound 4; right lower corner: (α-D-Mannopyranosyl)-(1.fwdarw.2)-(α-D-mannopyranosyl)-(1.fwdarw.2)-(α-D-mannopyranosyl)-(1.fwdarw.6)-(α-D-mannopyranosyl)-(1.fwdarw.4)-(2-amino-2-deoxy-α-D-glucopyranosyl)-(1.fwdarw.6)-1-O-(6-thiohexyl phosphono)-D-myo-inositol (Chem. Eur. J. 2005, 11, 2493). From the fluorescence pattern it can be seen that such specific antibodies were produced by the mouse that these antibodies specifically evoke a binding to compound 3, and not to the structurally very related compounds 4, 5 and (α-D-Mannopyranosyl)-(1.fwdarw.2)-(α-D-mannopyranosyl)-(1.fwdarw.2)-(α-D-mannopyranosyl)-(1.fwdarw.6)-(α-D-mannopyranosyl)-(1.fwdarw.4)-(2-amino-2-deoxy-α-D-glucopyranosyl)-(1.fwdarw.6)-1-O-(6-thiohexyl phosphono)-D-myo-inositol.

(4) FIG. 4: Fluorescence microscopic image of tachyzoiten stained with serum that was obtained after immunization of Balb/c mice with a conjugate consisting of compound 3 covalently linked to CRM.sub.197; blue: cell nucleus, DAPI; green: fluorescent secondary antibody. The fluorescence image shows that the mouse serum which was derived from a mouse immunized with a conjugate consisting of compound 3 covalently linked to CRM.sub.197 effectively binds to tachyzoites of T. gondii.

(5) FIG. 5: A) Possible functional groups X being attached to a suitable carrier; B) Possible reaction pathway of attaching a compound of the general formula (I) to a carrier modified with a vinyl functional group X by an thiol-ene reaction upon activation by irradiation of light and/or by a radical starter.

(6) FIG. 6: Preparation of conjugates of compounds 3 and 4 with CRM.sub.197 for immunization: a) maleimide-modification of CRM.sub.197 PBS, pH=7.4, room temperature, 2 h b) coupling of compounds 3 and 4: PBS, pH=7.4, room temperature, 3 h.

(7) FIG. 7: Selection of reactive bifunctional molecules A suitable for modifying a carrier material for subsequent introduction of a compound of the formula (I) on the carrier material by direct bonding.

(8) FIG. 8: Preparation of the conjugate of compound 4 with CRM.sub.197 for immunization: a) α-iodoacetamide modification of CRM.sub.197: PBS, pH=7.4, 1 h, room temperature; b) coupling of compound 4: compound 4, PBS, pH=8.5, 3 h.

(9) FIG. 9: MALDI-TOF analysis of A) CRM.sub.197 (blue, 58.5 kDa), B) CRM.sub.197-iodoacetamide (red, 68 kDa) and C) CRM.sub.197-GPI conjugate of compound 4 with CRM.sub.197 (black, 72 kDa); D) Comparison of the MALDI-TOF analyses of CRM.sub.197 (blue, 58.5 kDa), CRM.sub.197-iodoacetamide (red, 68 kDa) and CRM.sub.197-GPI conjugate of compound 4 with CRM.sub.197 (black, 72 kDa). As a Matrix 2′,4′,6′-Trihydroxyacetophenone (THAP) was used.

(10) FIG. 10: In flow preparation of the conjugate of compound 4 with a vinyl-modified glycosphingolipid with immunomodulatory properties.

(11) FIG. 11: Serum antibody levels against compound 4 in mice immunized with the conjugate obtained as described in example 11e: A) Total serum IgG levels; B) IgG subclass levels; Bars represent mean values averaged over all mice including standard error of the mean; FI=fluorescence intensity.

(12) FIG. 12: Specificity and epitope recognition of the antibody response: Pictures of microarrays incubated with serum (dilution 1:1000) of the three mice six weeks after 1.sup.st immunization and a secondary fluorescent antibody directed against mouse IgG. Compound 4 as well as the shown substructures were printed at 100 μM.

(13) FIG. 13: Recognition of the natural GPI antigen displayed on the T. gondii parasite by the antibodies raised against compound 4: IF pictures of paraformaldehyde-fixed purified T. gondii tachyzoites grown in human foreskin fibroblasts stained with (A) DAPI (B) pooled serum from immunized mice and a secondary FITC-conjugated anti-mouse-IgG (C) differential interference contrast picture and (D) merge of (A), (B) and (C). Full circle in (C) indicates the apical and half circle the basolateral end of the parasite (white bar=5 μm).

EXPERIMENTAL PART

(14) Part A1: Preparation of Phosphate Linked Thiol Functionalized GPI

Example 1: Triethylammonium 2,3,4-Tri-O-benzyl-6-O-triisopropylsilyl-α-D-mannopyranosyl-(1→2)-3,4,6-tri-O-benzyl-α-D-mannopyranosyl-(1→6)-4-O-(3,4,6-tri-O-benzyl-2-deoxy-2-acetamido-β-D-galactopyranosyl)-3-O-benzyl-2-O-levulinyl-α-D-mannopyranosyl-(1→4)-2-azido-3,6-di-O-benzyl-2-deoxy-α-D-glucopyranosyl-(1→6)-1-O-(6-(S-benzyl)thiohexyl phosphono)-2,3,4,5-tetra-O-benzyl-D-myo-inositol (Compound 12)

(15) ##STR00011##

(16) 2,3,4-Tri-O-benzyl-6-O-triisopropylsilyl-α-D-mannopyranosyl-(1.fwdarw.2)-3,4,6-tri-O-benzyl-α-D-mannopyranosyl-(1.fwdarw.6)-4-O-(3,4,6-tri-O-benzyl-2-deoxy-2-acetamido-β-D-galactopyranosyl)-3-O-benzyl-2-O-levulinyl-α-D-mannopyranosyl-(1.fwdarw.4)-2-azido-3,6-di-O-benzyl-2-deoxy-α-D-glucopyranosyl-(1.fwdarw.6)-1-O-allyl-2,3,4,5-tetra-O-benzyl-D-myo-inositol (compound 9) (20 mg, 7.26 μmol, 1 equiv) and triethylammonium 6-(benzylthio)hexyl phosphonate (compound 10) (12.7 mg, 33 μmol, 4.5 equiv) are co-evaporated 3 times with 2 mL dry pyridine. The residue is dissolved in 2 mL dry pyridine and PivCl (6.70 μL, 54 μmol, 7.5 equiv) is added. The solution is stirred for 2 h at r.t. before water (10 μL, 0.56 mmol, 76 equiv) and iodine (10.1 mg, 40 μmol, 5.5 equiv) are added. The red solution is stirred for 1 h and is quenched with sat. Na.sub.2S.sub.3O.sub.3. The reaction mixture is diluted with 10 mL CHCl3 and dried over Na.sub.2SO.sub.4. The solvents are removed in vacuo and the residue is purified through flash column chromatography (starting from CHCl3/MeOH 0%.fwdarw.5% MeOH) to yield yellow oil (18 mg, 5.9 μmol, 82%).

(17) [α].sub.D.sup.20=+32.6 (c=1.00 in CHCl.sub.3); ν.sub.max (neat) 2926, 2864, 2107, 1742, 1720, 1677, 1454, 1098, 1059, 1028 cm.sup.−1; .sup.1H NMR (600 MHz, CDCl.sub.3) δ 7.35 (d, J=7.4 Hz, 2H), 7.31-7.04 (m, 81H), 6.99 (dd, J=6.6, 2.8 Hz, 2H), 5.94 (d, J=8.8 Hz, 1H, NH), 5.86 (d, J=3.7 Hz, 1H, GlcNH.sub.2-1), 5.27-5.24 (m, 2H, ManI-2), 5.09 (d, J=1.2 Hz, 1H), 4.96 (d, J=12.0 Hz, 1H, CH.sub.2 of Bn), 4.92-4.81 (m, 3H, CH.sub.2 of Bn), 4.81-4.57 (m, 11H), 4.57-4.48 (m, 4H), 4.48-4.38 (m, 4H), 4.38-4.19 (m, 12H), 4.11-3.72 (m, 17H), 3.69 (dd, J=9.7, 7.0 Hz, 1H), 3.63-3.54 (m, 8H), 3.52 (t, J=6.4 Hz, 1H), 3.50-3.30 (m, 8H), 3.25 (dd, J=7.8, 3.9 Hz, 2H), 3.05 (dd, J=10.2, 3.7 Hz, 1H, GlcNH.sub.2-2), 2.86 (q, J=7.3 Hz, 6H, NCH.sub.2CH.sub.3), 2.28 (t, 2H, —S—CH.sub.2—CH.sub.2—CH.sub.2—CH.sub.2—CH.sub.2—CH.sub.2—O), 2.23-2.08 (m, 4H, CH.sub.2 of Lev), 1.83 (s, 3H, NHAc), 1.56-1.48 (m, 5H, CH.sub.3 of Lev, —S—CH.sub.2—CH.sub.2—CH.sub.2—CH.sub.2—CH.sub.2—CH.sub.2—O), 1.42 (m.sub.centered, 2H, —S—CH.sub.2—CH.sub.2—CH.sub.2—CH.sub.2—CH.sub.2—CH.sub.2—O), 1.26-1.14 (m, 13H, NCH.sub.2CH.sub.3, —S—CH.sub.2—CH.sub.2—CH.sub.2—CH.sub.2—CH.sub.2—CH.sub.2—O), 1.00-0.93 (m, 21H, TIPS); .sup.13C NMR (151 MHz, CDCl.sub.3) δ 205.92 (ketone of Lev), 171.77 (CO of Lev), 170.35 (CONN), 140.05, 139.05, 138.95, 138.88, 138.83, 138.75, 138.68, 138.58, 138.57, 138.40, 138.33, 138.24, 138.05, 137.99, 137.71, 128.92, 128.64, 128.62, 128.59, 128.57, 128.54, 128.46, 128.42, 128.40, 128.36, 128.32, 128.28, 128.26, 128.21, 128.14, 128.11, 128.09, 128.08, 128.00, 127.96, 127.84, 127.77, 127.72, 127.68, 127.67, 127.61, 127.56, 127.51, 127.49, 127.44, 127.28, 127.14, 126.97, 126.94, 100.58, 99.48, 98.79, 98.66, 96.51 (GlcNH.sub.2-1), 81.99, 81.75, 81.23, 80.89, 80.52, 79.77, 79.24, 76.01, 75.76, 75.73, 75.57, 75.44, 75.34, 75.13, 74.76, 74.57, 74.40, 74.37, 74.26, 74.24, 74.20, 73.93, 73.85, 73.62, 73.27, 73.15, 72.96, 72.68, 72.40, 72.37, 72.32, 72.28, 72.09, 71.57, 71.49, 71.07, 70.77, 69.95 (ManI-2), 69.76, 68.98, 68.74, 66.74, 65.73, 65.69, 63.71 (GlcNH.sub.2-2), 62.94, 53.25, 45.58, 37.90, 36.38, 31.46, 31.07, 31.02, 29.69, 29.30, 28.82, 28.06, 25.55, 23.22, 18.24, 18.18, 12.17, 8.74; .sup.31P NMR (162 MHz, CDCl.sub.3) δ −0.30; m/z (ESI) Found: [M+Na].sup.+, 3062.3573; C.sub.177H.sub.205N4O.sub.35PSSi requires [M+Na].sup.+, 3062.3577.

Example 2: Triethylammonium 2,3,4-Tri-O-benzyl-α-D-mannopyranosyl-(1→2)-3,4,6-tri-O-benzyl-α-D-mannopyranosyl-(1→6)-4-O-(3,4,6-tri-O-benzyl-2-deoxy-2-acetamido-β-D-galactopyranosyl)-3-O-benzyl-2-O-levulinyl-α-D-mannopyranosyl-(1→4)-2-azido-3,6-ucopyranosyl-O-benzyl-2-deoxy-α-D-glucopyranosyl-(1→6)-1-O-(6-(S-benzyl)thiohexyl phosphono)-2,3,4,5-tetra-O-benzyl-D-myo-inositol (compound 18)

(18) ##STR00012##

(19) Triethylammonium 2,3,4-Tri-O-benzyl-6-O-triisopropylsilyl-α-D-mannopyranosyl-(1.fwdarw.2)-3,4,6-tri-O-benzyl-α-D-mannopyranosyl-(1.fwdarw.6)-4-O-(3,4,6-tri-O-benzyl-2-deoxy-2-acetamido-β-D-galactopyranosyl)-3-O-benzyl-2-O-levulinyl-α-D-mannopyranosyl-(1.fwdarw.4)-2-azido-3,6-di-O-benzyl-2-deoxy-α-D-glucopyranosyl-(1.fwdarw.6)-1-O-(6-(S-benzyl)thiohexyl phosphono)-2,3,4,5-tetra-O-benzyl-D-myo-inositol (compound 12) (59 mg, 19 μmol, 1 equiv) is dissolved in 2 mL MeCN. Water (13.5 μL, 0.75 mmol, 40 equiv) and Sc(TfO).sub.3 (18.5 mg, 38 μmol, 2 equ.) are added and the solution is heated up to 50° C. for 5 h. The reaction is quenched with pyridine (7.6 μL, 94 μmol, 5 equiv) and the solvents are removed in vacuo. The residue is purified through flash column chromatography (starting from CHCl3/MeOH 0%.fwdarw.5% MeOH) to yield colorless oil (52 mg, 18 μmol, 93%).

(20) [α].sub.D.sup.20=+31.3 (c=1.10 in CHCl.sub.3); ν.sub.max (neat) 3346, 2925, 2107, 1742, 1719, 1669, 1497, 1454, 1362, 1048, 912 cm.sup.−1; .sup.1H NMR (600 MHz, CDCl.sub.3) δ 7.43-6.88 (m, 85H), 6.01 (d, J=8.2 Hz, 1H, NH), 5.88 (d, J=3.7 Hz, 1H, GlcNH.sub.2-1), 5.27-5.14 (m, 2H, ManI-2), 4.94 (m, 1H), 4.90-4.63 (m, 13H), 4.59 (d, J=10.7 Hz, 1H), 4.55-4.28 (m, 18H), 4.25-4.20 (m, 1H), 4.17 (dd, J=11.8, 5.3 Hz, 2H), 4.13-3.97 (m, 3H), 3.95 (t, J=2.2 Hz, 1H), 3.90 (t, J=9.6 Hz, 1H), 3.87-3.50 (m, 23H), 3.49-3.38 (m, 7H), 3.16 (dd, J=6.9, 3.1 Hz, 1H), 3.06 (dd, J=10.2, 3.7 Hz, 1H, GlcNH.sub.2-2), 2.76 (q, J=7.2 Hz, 6H, NCH.sub.2CH.sub.3), 2.34-2.08 (m, 6H, CH.sub.2 of Lev, —S—CH.sub.2—CH.sub.2—CH.sub.2—CH.sub.2—CH.sub.2—CH.sub.2—O), 1.85 (s, 3H, CH.sub.3 of Lev), 1.64 (s, 3H, NHAc), 1.54-1.47 (m, 2H, —S—CH.sub.2—CH.sub.2—CH.sub.2—CH.sub.2—CH.sub.2—CH.sub.2—O), 1.45-1.37 (m, 2H, —S—CH.sub.2—CH.sub.2—CH.sub.2—CH.sub.2—CH.sub.2—CH.sub.2—O), 1.25-1.16 (m, 4H, —S—CH.sub.2—CH.sub.2—CH.sub.2—CH.sub.2—CH.sub.2—CH.sub.2—O), 1.13 (t, J=7.3 Hz, 9H, NCH.sub.2CH.sub.3); .sup.13C NMR (151 MHz, CDCl.sub.3) δ 206.17 (ketone of Lev), 171.72, 170.70, 140.03, 139.03, 138.87, 138.74, 138.69, 138.65, 138.62, 138.52, 138.48, 138.44, 138.41, 138.35, 138.19, 138.10, 138.00, 128.91, 128.63, 128.54, 128.53, 128.47, 128.45, 128.39, 128.36, 128.34, 128.33, 128.30, 128.28, 128.25, 128.11, 128.09, 128.08, 128.02, 128.00, 127.96, 127.81, 127.81, 127.77, 127.73, 127.67, 127.60, 127.56, 127.52, 127.47, 127.45, 127.43, 127.34, 127.24, 127.12, 126.93, 100.75, 99.86, 99.32, 98.90, 96.39 (GlcNH.sub.2-1), 81.92, 81.79, 81.18, 80.09, 80.05, 79.55, 79.07, 76.08, 75.92, 75.72, 75.41, 75.26, 75.14, 75.10, 75.05, 74.94, 74.73, 74.55, 74.10, 73.95, 73.52, 73.48, 73.43, 73.09, 73.03, 72.42, 72.36, 72.29, 72.23, 72.18, 71.72, 71.28, 69.97, 69.67, 69.46 (ManI-2), 68.90, 68.67, 67.15, 65.71, 65.67, 63.39 (Glc-NH.sub.2-2), 62.37, 54.27, 45.89 (NCH.sub.2CH.sub.3), 37.88, 36.36, 31.45 (—S—CH.sub.2—CH.sub.2—CH.sub.2—CH.sub.2—CH.sub.2—CH.sub.2—O), 31.06, 31.01, 29.70, 29.29, 28.81, 28.01, 25.54, 23.44, 9.62 (NCH.sub.2CH.sub.3); .sup.31P NMR (243 MHz, CDCl.sub.3) δ −1.08; m/z (ESI) Found: [M+Na].sup.+, 2922.2032; C.sub.168H.sub.185N.sub.4O.sub.35PSSi requires [M+Na].sup.+, 2922.1969.

Example 3: Bistriethylammonium 2,3,4-Tri-O-benzyl-6-O-(2-(N-benzyloxy carbonyl) aminoethyl phosphono)-α-D-mannopyranosyl-(1→2)-3,4,6-tri-O-benzyl-α-D-mannopyranosyl-(1→6)-4-O-(3,4,6-tri-O-benzyl-2-deoxy-2-acetamido-β-D-galactopyranosyl)-3-O-benzyl-α-D-mannopyranosyl-(1→4)-2-azido-3,6-di-O-benzyl-2-deoxy-α-D-glucopyranosyl-(1→6)-1-O-(6-(S-benzyl)thiohexyl phosphono)-2,3,4,5-tetra-O-benzyl-D-myo-inositol (compound 15)

(21) ##STR00013##

(22) Triethylammonium 2,3,4-Tri-O-benzyl-α-D-mannopyranosyl-(1.fwdarw.2)-3,4,6-tri-O-benzyl-α-D-mannopyranosyl-(1.fwdarw.6)-4-O-(3,4,6-tri-O-benzyl-2-deoxy-2-acetamido-β-D-galactopyranosyl)-3-O-benzyl-2-O-levulinyl-α-D-mannopyranosyl-(1.fwdarw.4)-2-azido-3,6-di-O-benzyl-2-deoxy-α-D-glucopyranosyl-(1.fwdarw.6)-1-O-(6-(S-benzypthiohexyl phosphono)-2,3,4,5-tetra-O-benzyl-D-myo-inositol (compound 18) (28.3 mg, 78 μmol, 4.5 equiv) and triethylammonium 2-(((benzyloxy)carbonyl)amino)ethyl phosphonate (compound 13) (28.3 mg, 78 μmol, 4.5 equiv) are co evaporated 3 times with 2 mL dry pyridine. The residue is dissolved in 2 mL dry pyridine and PivCl (16.1 μL, 131 μmol, 7.5 equiv) is added. The solution is stirred for 2 h at r.t. before water (15.6 μL, 0.87 mmol, 50 equiv) and iodine (24.3 mg, 96 μmol, 5.5 equiv) are added. The red solution is stirred for 1 h and is quenched with hydrazine (1M in THF; 300 μL, 0.3 mmol, 17 equiv). The reaction mixture is stirred for 18 h. The solvents are removed in vacuo and the residue is purified through flash column chromatography (starting from CHCl3/MeOH: 97/3490/10) to yield yellow oil (49.5 mg, 15 μmol, 88%).

(23) [α].sub.D.sup.20=+32.5 (c=1.00 in CHCl.sub.3); ν.sub.max (neat) 3387, 3063, 2929, 2108, 1672, 1497, 1057, 1029, 839 cm.sup.−1; .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.37-6.90 (m, 90H), 6.28 (s, 1H, NHAc), 5.89 (d, J=3.5 Hz, 1H, GlcNH.sub.2-1), 5.19 (d, J=1.6 Hz, 1H), 5.05-4.15 (m, 40H), 4.14-4.02 (m, 3H), 3.98-3.35 (m, 36H), 3.28-3.20 (m, 1H), 3.14 (dd, J=9.2, 4.5 Hz, 1H), 3.05 (dd, J=10.2, 3.5 Hz, 1H, GlcNH.sub.2-2), 2.61 (q, J=7.3 Hz, 12H, NCH.sub.2CH.sub.3), 2.28 (t, J=7.4 Hz, 2H, —S—CH.sub.2—CH.sub.2—CH.sub.2—CH.sub.2—CH.sub.2—CH.sub.2—O), 1.92-1.76 (m, 3H, COCH.sub.3), 1.58-1.37 (m, 4H, —S—CH.sub.2—CH.sub.2—CH.sub.2—CH.sub.2—CH.sub.2—CH.sub.2—O), 1.26-1.13 (m, 4H, —S—CH.sub.2—CH.sub.2—CH.sub.2—CH.sub.2—CH.sub.2—CH.sub.2—O), 0.97 (t, J=7.3 Hz, 18H, NCH.sub.2CH.sub.3); .sup.13C NMR (151 MHz, CDCl.sub.3) δ 170.84 (COCH.sub.3), 156.58 (O(CO)NH), 138.71, 128.87, 128.50, 128.44, 128.36, 128.30, 128.21, 128.03, 127.94, 127.86, 127.76, 127.50, 126.91, 102.05, 101.15, 100.26, 98.71, 96.19, 82.02, 81.79, 81.06, 80.17, 80.03, 79.66, 77.37, 77.16, 76.95, 76.36, 75.93, 75.58, 75.19, 74.84, 74.80, 74.65, 74.03, 73.49, 72.79, 72.29, 71.62, 71.56, 71.49, 71.41, 70.08, 69.75, 69.64, 68.97, 66.34, 65.70, 65.04, 64.09, 63.49, 58.17, 45.70, 38.76, 36.34, 32.00, 31.44, 29.78, 29.59, 29.44, 29.27, 28.80, 27.54, 25.50, 22.77, 14.21, 8.71; .sup.31P NMR (243 MHz, CDCl.sub.3) δ−0.03, −1.67; m/z (ESI) Found: [M-H].sup.−, 3041.2393; C.sub.173H.sub.191N.sub.5O.sub.38P.sub.2S requires [M-H].sup.−, 3041.2353.

Example 4: 6-O-(aminoethyl phosphono)-α-D-mannopyranosyl-(1→2)-α-D-mannopyranosyl-(1→6)-4-O-(2-deoxy-2-acetamido-β-D-galactopyranosyl)-α-D-mannopyranosyl-(1→4)-2-amino-2-deoxy-α-D-glucopyranosyl-(1→6)-1-O-(thiohexyl phosphono)-D-myo-inositol (Compound 3)

(24) ##STR00014##

(25) Approximately 10 mL ammonia were condensed in a flask and tert-BuOH (2 drops) was added. Afterwards small pieces of sodium were added till a dark blue colour was established. Bistriethylammonium 2,3,4-Tri-O-benzyl-6-O-(2-(N-benzyloxycarbonyl)aminoethyl phosphono)-α-D-mannopyranosyl-(1.fwdarw.2)-3,4,6-tri-O-benzyl-α-D-mannopyranosyl-(1.fwdarw.6)-4-O-(3,4,6-tri-O-benzyl-2-deoxy-2-acetamido-β-D-galactopyranosyl)-3-O-benzyl-α-D-mannopyranosyl-(1.fwdarw.4)-2-azido-3,6-di-O-benzyl-2-deoxy-α-D-glucopyranosyl-(1.fwdarw.6)-1-O-(6-(S-benzypthiohexyl phosphono)-2,3,4,5-tetra-O-benzyl-D-myo-inositol (compound 15) (28 mg, 8.6 μmol) was dissolved in dry THF (1.5 mL) and added to the ammonium solution at −78° C. The reaction was stirred for 30 min at this temperature. The reaction was quenched with dry MeOH (2 mL) and the ammonia was blown off using a stream of nitrogen. The pH of the resulting solution was adjusted with concentrated acetic acid to 8-9. Solvents were removed in vacuo and the residue was purified using a small G10 column (GE Healthcare) to yield X as white solid (6.7 mg, 4.9 μmol, 58%): .sup.1H NMR (600 MHz, D.sub.2O) δ 5.54 (d, J=3.9 Hz, 1H, GlcNH.sub.2), 5.23 (s, 1H), 5.19 (s, 1H), 5.03 (s, 1H), 4.51 (d, J=8.3 Hz, 1H, GalNAc-1), 4.29-3.66 (m, 36H), 3.63-3.53 (m, 2H), 3.45 (td, J=9.3, 4.3 Hz, 1H), 3.38 (dd, J=10.9, 4.3 Hz, 1H, GlcNH.sub.2-2), 3.34-3.29 (m, 2H), 2.80 (t, J=7.1 Hz, 1H), 2.58 (t, J=7.1 Hz, 1H), 2.12 (s, 3H, Me of NHAc), 1.80-1.59 (m, 4H, linker), 1.51-1.36 (m, 4H, linker); .sup.13C NMR (151 MHz, D.sub.2O) δ 177.30 (amide), 105.02, 104.34 (GalNAc-1), 104.09, 101.16, 98.15 (Glc-NH.sub.2-1), 81.69, 79.37, 78.78, 78.01, 75.69, 75.40, 74.84, 74.60, 74.05, 73.92, 73.62, 73.15, 73.08, 72.82, 72.64, 72.49, 72.03, 71.56, 70.32, 69.61, 69.21, 68.85, 67.28, 64.53, 64.50, 63.76, 63.70, 62.81, 56.64 (GlcNH.sub.2-2), 55.19, 42.66, 40.73, 35.53, 32.31, 30.88, 29.67, 27.20, 27.00, 26.29, 24.96 (Me of NHAc); .sup.31P NMR (243 MHz, D.sub.2O) δ −2.62, −2.83; m/z (ESI) Found: [M−2H].sup.2−, 673.7104; C.sub.46H.sub.85N.sub.3O.sub.36P.sub.2S requires [M−2H].sup.2−, 673.6981.

Example 5: Tristriethylammonium 2,3,4-Tri-O-benzyl-6-O-(2-(N-benzyloxycarbonyl)aminoethyl phosphono)-α-D-mannopyranosyl-(1→2)-3,4,6-tri-O-benzyl-α-D-mannopyranosyl-(1→6)-4-O-(3,4,6-tri-O-benzyl-2-deoxy-2-acetamido-β-D-galactopyranosyl)-2-(2-(N-benzyloxycarbonyl)aminoethyl phosphono)-3-O-benzyl-α-D-mannopyranosyl-(1→4)-2-azido-3,6-di-O-benzyl-2-deoxy-α-D-glucopyranosyl-(1→6)-1-O-(6-(S-benzyl)thiohexyl phosphono)-2,3,4,5-tetra-O-benzyl-D-myo-inositol (Compound 16)

(26) ##STR00015##

(27) Bistriethylammonium 2,3,4-Tri-O-benzyl-6-O-(2-(N-benzyloxycarbonyl) aminoethyl phosphono)-α-D-mannopyranosyl-(1.fwdarw.2)-3,4,6-tri-O-benzyl-α-D-mannopyranosyl-(1.fwdarw.6)-4-O-(3,4,6-tri-O-benzyl-2-deoxy-2-acetamido-β-D-galactopyranosyl)-3-O-benzyl-α-D-mannopyranosyl-(1.fwdarw.4)-2-azido-3,6-di-O-benzyl-2-deoxy-α-D-glucopyranosyl-(1.fwdarw.6)-1-O-(6-(S-benzyl)thiohexyl phosphono)-2,3,4,5-tetra-O-benzyl-D-myo-inositol (compound 15) (15 mg, 4.6 μmol, 1 equiv) and triethylammonium 2-(((benzyloxy)carbonyl)amino)ethyl phosphonate (compound 13) (8.1 mg, 22.5 μmol, 4.5 equiv) are co evaporated 3 times with 2 mL dry pyridine. The residue is dissolved in 2 mL dry pyridine and PivCl (4.6 μL, 36.8 μmol, 7.5 equiv) is added. The solution is stirred for 2 h at r.t. before water (10 μL, 0.56 mmol, 76 equiv) and iodine (6.8, 27 μmol, 5.5 equiv) are added. The red solution is stirred for 1 h and is quenched with sat. Na.sub.2S.sub.3O.sub.3. The reaction mixture is diluted with 10 mL CHCl3 and dried over Na.sub.2SO.sub.4. The solvents are removed in vacuo and the residue is purified through flash column chromatography (CHCl3/MeOH 100/0480/20) to yield yellow oil (13.5 mg, 3.8 μmol, 76%).

(28) [α].sub.D.sup.20=+22.0 (c=1.00 in CHCl.sub.3); ν.sub.max (neat) 3358, 2927, 2108, 1641, 1454, 1398, 1054, 7028, 838, 804 cm.sup.−1; .sup.1H NMR (600 MHz, CDCl.sub.3) δ 7.56-6.77 (m, 90H), 6.44 (s, 2H, CbzNH), 6.22 (s, 1H, NHCOCH.sub.3), 5.87 (s, 1H, GlcNH.sub.2-1), 5.49 (s, 1H), 5.09-3.35 (m, 83H), 3.30 (dd, J=14.2, 7.1 Hz, 1H), 3.26-3.17 (m, 2H), 3.17-3.08 (m, 2H), 3.07-2.79 (m, 1H), 2.58 (q, J=7.2 Hz, 18H, NCH.sub.2CH.sub.3), 2.27 (t, J=7.4 Hz, 2H, —S—CH.sub.2—CH.sub.2—CH.sub.2—CH.sub.2—CH.sub.2—CH.sub.2—O), 2.05-1.98 (m, 2H), 1.90-1.83 (m, 3H, NHCOCH.sub.3), 1.59-1.36 (m, 4H, —S—CH.sub.2—CH.sub.2—CH.sub.2—CH.sub.2—CH.sub.2—CH.sub.2—O), 1.27-1.12 (m, 4H, —S—CH.sub.2—CH.sub.2—CH.sub.2—CH.sub.2—CH.sub.2—CH.sub.2—O), 0.99 (t, J=7.2 Hz, 27H, NCH.sub.2CH.sub.3); .sup.13C NMR (151 MHz, CDCl.sub.3) δ 156.57 (OCONH), 140.07, 139.03, 138.78, 138.73, 138.67, 138.32, 137.23, 128.93, 128.54, 128.49, 128.44, 128.39, 128.36, 128.31, 128.28, 128.25, 128.23, 128.18, 128.03, 128.01, 127.97, 127.93, 127.82, 127.71, 127.60, 127.57, 127.54, 127.51, 127.41, 127.38, 127.30, 127.15, 127.06, 126.94, 126.84, 100.59, 98.53, 96.54 (GlcNH.sub.2-1), 81.89, 81.20, 75.58, 75.04, 74.83, 74.67, 73.28, 72.84, 72.30, 66.39, 66.23, 65.70, 63.97, 45.85 (NCH.sub.2CH.sub.3), 42.97, 42.52, 40.10, 36.39, 34.58, 33.94, 32.05, 31.56, 31.50 (S—CH.sub.2—CH.sub.2—CH.sub.2—CH.sub.2—CH.sub.2—CH.sub.2—O), 31.08, 31.03, 30.33, 29.82, 29.79, 29.74, 29.63, 29.48, 29.32, 29.28, 29.08, 28.84, 25.55, 22.81, 21.56, 14.99, 14.30, 14.24, 13.23, 9.91 (NCH.sub.2CH.sub.3); .sup.31P NMR (243 MHz, CDCl.sub.3) δ 0.17, −0.02, −1.15; m/z (ESI) Found: [M+5Na−3H].sup.2+, 1705.6285; C.sub.183H.sub.203N.sub.6O.sub.43P.sub.3S requires [M+5Na−3H].sup.2+, 1705.6062.

Example 6: 6-O-(aminoethyl phosphono)-α-D-mannopyranosyl-(1→2)-α-D-mannopyranosyl-(1→6)-2-O-(aminoethyl phosphono)-4-O-(2-deoxy-2-acetamido-β-D-galactopyranosyl)-α-D-mannopyranosyl-(1→4)-2-amino-2-deoxy-α-D-glucopyranosyl-(1→6)-1-O-(thiohexyl phosphono)-D-myo-inositol (Compound 5)

(29) ##STR00016##

(30) Tristriethylammonium 2,3,4-Tri-O-benzyl-6-O-(2-(N-benzyloxycarbonyl) aminoethyl phosphono)-α-D-mannopyranosyl-(1.fwdarw.2)-3,4,6-tri-O-benzyl-α-D-mannopyranosyl-(1.fwdarw.6)-4-O-(3,4,6-tri-O-benzyl-2-deoxy-2-acetamido-β-D-galactopyranosyl)-2-(2-(N-benzyloxycarbonyl)aminoethyl phosphono)-3-O-benzyl-α-D-mannopyranosyl-(1.fwdarw.4)-2-azido-3,6-di-O-benzyl-2-deoxy-α-D-glucopyranosyl-(1.fwdarw.6)-1-O-(6-(S-benzyl)thiohexyl phosphono)-2,3,4,5-tetra-O-benzyl-D-myo-inositol (compound 16) (22 mg, 6.1 μmol, 1 equiv) was dissolved in dry THF (15 mL) and dry tert-BuOH (0.1 mL). The solution is cooled down to −78° C. and approximately 20 mL ammonia is condensed in the flask. Afterwards small pieces of sodium are added. The solution is warmed to about −40° C. till a dark blue colour is established. Then the solution is cooled down to −78° C. and the reaction is stirred for 1 h at this temperature. The reaction is quenched with 2 mL dry MeOH and the ammonia is blown off of using a stream of nitrogen. Solvents are afterwards evaporated and the residue is dissolved in 5 mL water. The pH of the solution is adjusted with concentrated acetic acid to 4-7. Water is removed by freeze drying and the residue is purified using a small G25 column (1 cm×20 cm) to yield a white solid (2.6 mg, 1.8 μmol, 29%).

(31) .sup.1H NMR (400 MHz, D.sub.2O) δ 5.57-5.52 (m, 1H, GlcNH.sub.2-1), 5.45 (s, 1H, ManI-1), 5.19 (s, 1H), 5.04 (s, 1H), 4.53 (d, J=8.4 Hz, 2H, GalNAc-1, ManI-2), 4.29-3.64 (m, 37H), 3.62-3.53 (m, 2H), 3.44 (t, J=9.3 Hz, 1H), 3.41-3.34 (m, 1H), 3.34-3.26 (m, 4H), 2.79 (t, J=7.3 Hz, 2H, HS—CH.sub.2), 2.11 (s, 3H, Me of NHAc), 1.81-1.57 (m, 4H), 1.53-1.36 (m, 4H).; .sup.31P NMR (162 MHz, D.sub.2O) δ 0.36, 0.14, −0.81; m/z (ESI) Found: [M−3H].sup.−3, 979.96; C.sub.96H.sub.180N.sub.8O.sub.78P.sub.6S.sub.2 requires [M−3H].sup.−3, 979.93.

Example 7: Triethylammonium 2,3,4-Tri-O-benzyl-6-O-triisopropylsilyl-α-D-mannopyranosyl-(1→2)-3,4,6-tri-O-benzyl-α-D-mannopyranosyl-(1→6)-3-O-benzyl-4-O-(2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl-(1→4)-3,6-di-O-benzyl-2-deoxy-2-acetamido-β-D-galactopyranosyl)-2-O-levulinyl-α-D-manno-pyranosyl-(1→4)-2-azido-3,6-di-O-benzyl-2-deoxy-α-D-glucopyranosyl-(1→6)-1-O-(6-(S-benzyl)thiohexyl phosphono)-2,3,4,5-tetra-O-benzyl-D-myo-inositol (Compound 11)

(32) ##STR00017##

(33) 2,3,4-Tri-O-benzyl-6-O-triisopropylsilyl-α-D-mannopyranosyl-(1.fwdarw.2)-3,4,6-tri-O-benzyl-α-D-mannopyranosyl-(1.fwdarw.6)-3-O-benzyl-4-O-(2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl-(1.fwdarw.4)-3,6-di-O-benzyl-2-deoxy-2-acetamido-β-D-galactopyranosyl)-2-O-levulinyl-α-D-manno-pyranosyl-(1.fwdarw.4)-2-azido-3,6-di-O-benzyl-2-deoxy-α-D-glucopyranosyl-(1.fwdarw.6)-2,3,4,5-tetra-O-benzyl-D-myo-inositol (compound 8) (50 mg, 16 μmol, 1 equiv) and triethylammonium 6-(benzylthio)hexyl phosphonate (compound 10) (39 mg, 100 μmol, 6.4 equ.) are co evaporated 3 times with 2 mL dry pyridine. The residue is dissolved in 2 mL dry pyridine and PivCl (14.5 μL, 118 μmol, 7.5 equiv) is added. The solution is stirred for 2 h at r.t. before water (14 μL, 0.79 mmol, 50 equiv) and iodine (29.9 mg, 118 μmol, 7.5 equiv) are added. The red solution is stirred for 1 h and is quenched with sat. Na.sub.2S.sub.3O.sub.3. The reaction mixture is diluted with 10 mL CHCl3 and dried over Na.sub.2SO.sub.4. The solvents are removed in vacuo and the residue is purified through flash column chromatography (CHCl.sub.3/MeOH 100/0495/5) to yield yellow oil (49 mg, 14 μmol, 87%).

(34) [α].sub.D.sup.20=+42.3 (c=1.00 in CHCl.sub.3); ν.sub.max (neat) 3064, 3032, 2926, 2865, 2107, 1742, 1720, 1678, 1497, 1454, 1362, 1054, 1028, 913 cm.sup.−1; .sup.1H NMR (600 MHz, CDCl.sub.3) δ 7.49-6.87 (m, 100H), 6.07 (d, J=9.0 Hz, 1H, NH), 5.78 (s, 1H), 5.25 (s, 2H, ManI-2), 5.07 (s, 1H), 4.94 (d, J=11.9 Hz, 1H, CH.sub.2 of Bn), 4.89-3.89 (m, 49H), 3.86-3.74 (m, 11H), 3.73-3.66 (m, 2H), 3.63 (dd, J=8.9, 2.9 Hz, 1H), 3.60-3.45 (m, 11H), 3.44-3.36 (m, 4H), 3.34-3.22 (m, 5H), 3.03 (dd, J=10.1, 3.6 Hz, 1H), 2.85 (q, J=7.3 Hz, 6H, NCH.sub.2CH.sub.3), 2.78 (d, J=9.4 Hz, 1H), 2.27 (t, J=7.4 Hz, 2H, BnS—CH.sub.2), 2.24-2.04 (m, 4H, CH.sub.2 of Lev), 1.80 (s, 3H, CH.sub.3 of Lev), 1.70 (s, 3H, NHCOCH.sub.3), 1.55-1.34 (m, 4H, —S—CH.sub.2—CH.sub.2—CH.sub.2—CH.sub.2—CH.sub.2—CH.sub.2—O), 1.23-1.16 (m, 4H, —S—CH.sub.2—CH.sub.2—CH.sub.2—CH.sub.2—CH.sub.2—CH.sub.2—O), 1.12 (t, J=7.6 Hz, 9H, NCH.sub.2CH.sub.3), 1.00-0.94 (m, 21H, TIPS); .sup.13C NMR (151 MHz, CDCl.sub.3) δ 205.92 (ketone of Lev), 171.76 (CO of Lev), 170.21 (NHCOCH.sub.3), 140.02, 139.09, 139.03, 138.94, 138.91, 138.84, 138.76, 138.74, 138.69, 138.60, 138.57, 138.30, 138.15, 138.10, 138.01, 137.54, 128.92, 128.77, 128.72, 128.65, 128.62, 128.57, 128.54, 128.46, 128.44, 128.39, 128.36, 128.33, 128.31, 128.28, 128.27, 128.25, 128.24, 128.22, 128.16, 128.12, 128.10, 128.07, 127.98, 127.87, 127.84, 127.82, 127.77, 127.71, 127.67, 127.65, 127.63, 127.57, 127.55, 127.52, 127.48, 127.44, 127.29, 127.27, 127.18, 127.14, 126.94, 126.45, 101.71, 100.84, 99.65, 98.84, 98.65, 96.71, 82.20, 81.99, 81.51, 81.19, 81.03, 80.51, 80.12, 79.80, 79.13, 77.95, 77.53, 76.81, 75.93, 75.74, 75.43, 75.35, 75.26, 75.15, 74.75, 74.69, 74.54, 74.43, 74.03, 73.86, 73.63, 73.48, 73.23, 73.20, 73.00, 72.85, 72.39, 72.36, 72.07, 71.53, 71.37, 70.88, 70.79, 70.06 (ManI-2), 69.67, 68.77, 68.59, 67.74, 66.87, 65.80, 63.72, 62.90, 52.87, 45.42 (NCH.sub.2CH.sub.3), 38.57, 37.83, 36.38, 31.46, 31.00, 30.95, 29.82, 29.66, 29.30 (CH.sub.3 of Lev), 28.81, 28.04, 27.69, 27.41, 25.52, 23.31 (NHCOCH.sub.3), 18.24, 18.18, 14.25, 12.18, 8.55 (NCH.sub.2CH.sub.3); .sup.31P NMR (243 MHz, CDCl.sub.3) δ −1.45; m/z (ESI) Found: [M−2H].sup.2−, 1734.7564; C.sub.204H.sub.233N.sub.4O.sub.40PSSi requires [M−2H].sup.2−, 1734.7730.

Example 8: Triethylammonium 2,3,4-Tri-O-benzyl-α-D-mannopyranosyl-(1→2)-3,4,6-tri-O-benzyl-α-D-mannopyranosyl-(1→6)-3-O-benzyl-4-O-(2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl-(1→4)-3,6-di-O-benzyl-2-deoxy-2-acetamido-β-D-galactopyranosyl)-2-O-levulinyl-α-D-manno-pyranosyl-(1→4)-2-azido-3,6-di-O-benzyl-2-deoxy-α-D-glucopyranosyl-(1→6)-1-O-(6-(S-benzyl)thiohexyl phosphono)-2,3,4,5-tetra-O-benzyl-D-myo-inositol (Compound 17)

(35) ##STR00018##

(36) Triethylammonium 2,3,4-Tri-O-benzyl-6-O-triisopropylsilyl-α-D-mannopyranosyl-(1.fwdarw.2)-3,4,6-tri-O-benzyl-α-D-mannopyranosyl-(1.fwdarw.6)-3-O-benzyl-4-O-(2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl-(1.fwdarw.4)-3,6-di-O-benzyl-2-deoxy-2-acetamido-β-D-galactopyranosyl)-2-O-levulinyl-α-D-manno-pyranosyl-(1.fwdarw.4)-2-azido-3,6-di-O-benzyl-2-deoxy-α-D-glucopyranosyl-(1.fwdarw.6)-1-O-(6-(S-benzyl)thiohexyl phosphono)-2,3,4,5-tetra-O-benzyl-D-myo-inositol (compound 11) (43 mg, 12 μmol, 1 equiv) is dissolved in 2 mL MeCN. Water (8.7 μL, 0.48 mmol, 40 equiv) and Sc(TfO).sub.3 (11.8 mg, 24 μmol, 2 equiv) are added and the solution is heated up to 50° C. for 5 h. The reaction is quenched with pyridine (4.8 μL, 60 μmol, 5 equiv) and the solvents are removed in vacuo. The residue is purified through flash column chromatography (CHCl.sub.3/MeOH 100/0.fwdarw.95/5) to yield colorless oil (32 mg, 9.4 μmol, 78%).

(37) [a].sub.D.sup.20=+47.4 (c=1.00 in CHCl.sub.3); ν.sub.max (neat) 3363, 3031, 2926, 2862, 2107, 1742, 1719, 1671, 1497, 1454, 1362, 1068, 1049, 1028, 697 cm.sup.−1; .sup.1H NMR (600 MHz, CDCl.sub.3) δ 7.55-6.71 (m, 100H), 6.19 (s, 1H, NH), 5.87 (s, 1H), 5.20 (s, 1H), 5.17 (s, 1H), 5.03-4.16 (m, 40H), 4.15-3.33 (m, 43H), 3.28 (d, J=10.6 Hz, 1H), 3.21-3.11 (m, 1H), 3.02 (d, J=7.7 Hz, 1H), 2.87 (d, J=10.1 Hz, 1H), 2.80 (q, J=7.0 Hz, 6H, NCH.sub.2CH.sub.3), 2.35-2.13 (m, 6H, BnS—CH.sub.2, CH.sub.2 of Lev), 1.85 (s, 3H, CH.sub.3 of Lev), 1.75 (s, 3H, NHCOCH.sub.3), 1.59-1.35 (m, 4H, —S—CH.sub.2—CH.sub.2—CH.sub.2—CH.sub.2—CH.sub.2—CH.sub.2—O), 1.28-1.13 (m, 4H, —S—CH.sub.2—CH.sub.2—CH.sub.2—CH.sub.2—CH.sub.2—CH.sub.2—O), 1.10 (t, J=7.3 Hz, 1H, NCH.sub.2CH.sub.3);

(38) .sup.13C NMR (151 MHz, CDCl.sub.3) δ 206.20 (ketone of Lev), 171.68 (CO of Lev), 170.78 (NHCO), 149.97, 140.04, 139.04, 138.99, 138.87, 138.78, 138.68, 138.64, 138.54, 138.49, 138.30, 138.20, 138.10, 128.93, 128.82, 128.55, 128.49, 128.48, 128.43, 128.39, 128.37, 128.33, 128.30, 128.17, 128.08, 128.03, 127.96, 127.90, 127.80, 127.75, 127.66, 127.62, 127.59, 127.55, 127.53, 127.45, 127.35, 127.12, 126.95, 126.82, 101.45, 100.42, 100.04, 99.52, 99.19, 96.41, 82.15, 81.91, 81.21, 80.37, 79.99, 79.65, 77.96, 76.20, 75.86, 75.70, 75.36, 75.29, 75.13, 75.04, 74.93, 74.74, 74.10, 73.60, 73.53, 73.36, 73.29, 73.00, 72.56, 72.37, 72.31, 72.26, 72.17, 71.69, 71.20, 70.79, 69.84, 69.48, 69.26 (ManI-2), 69.00 67.98, 67.79, 65.75, 65.71, 63.32, 62.50, 54.82, 45.44 (NCH.sub.2CH.sub.3), 37.88, 36.39, 32.06, 31.47, 31.06, 29.91, 29.83, 29.72, 29.45, 29.39, 29.31, 28.84, 28.03, 27.71, 27.36, 25.55, 23.59, 22.83, 17.85, 14.26, 12.43, 8.60 (NCH.sub.2CH.sub.3); .sup.31P NMR (243 MHz, CDCl.sub.3) δ −1.12; m/z (ESI) Found: [M+Cl−H].sup.2−, 1673.6842; C.sub.195H.sub.213N.sub.4O.sub.40PS requires [M+Cl−H].sup.2−, 1673.6918.

Example 9: Bistriethylammonium 2,3,4-Tri-O-benzyl-6-O-(2-(N-benzyloxycarbonyl)aminoethyl phosphono)-α-D-mannopyranosyl-(1→2)-3,4,6-tri-O-benzyl-α-D-mannopyranosyl-(1→6)-3-O-benzyl-4-O-(2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl-(1→4)-3,6-di-O-benzyl-2-deoxy-2-acetamido-β-D-galactopyranosyl)-α-D-manno-pyranosyl-(1→4)-2-azido-3,6-di-O-benzyl-2-deoxy-α-D-glucopyranosyl-(1→6)-1-O-(6-(S-benzyl)thiohexyl phosphono)-2,3,4,5-tetra-O-benzyl-D-myo-inositol (Compound 14)

(39) ##STR00019##

(40) Triethylammonium 2,3,4-Tri-O-benzyl-α-D-mannopyranosyl-(1.fwdarw.2)-3,4,6-tri-O-benzyl-α-D-mannopyranosyl-(1.fwdarw.6)-3-O-benzyl-4-O-(2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl-(1.fwdarw.4)-3,6-di-O-benzyl-2-deoxy-2-acetamido-β-D-galactopyranosyl)-2-O-levulinyl-α-D-manno-pyranosyl-(1.fwdarw.4)-2-azido-3,6-di-O-benzyl-2-deoxy-α-D-glucopyranosyl-(1.fwdarw.6)-1-O-(6-(S-benzyl)thiohexyl phosphono)-2,3,4,5-tetra-O-benzyl-D-myo-inositol (compound 17) (31 mg, 9.1 μmol, 1 equiv) and triethylammonium 2-(((benzyloxy)carbonyl)amino)ethyl phosphonate (compound 13) (14.7 mg, 41 μmol, 4.5 equiv) are co evaporated 3 times with 2 mL dry pyridine. The residue is dissolved in 2 mL dry pyridine and PivCl (8.4 μL, 68 μmol, 7.5 equiv) is added. The solution is stirred for 2 h at r.t. before water (8.2 μL, 0.45 mmol, 50 equiv) and iodine (12.7 mg, 50 μmol, 5.5 equiv) are added. The red solution is stirred for 1 h and is quenched with hydrazine (1M in THF, 227 μL, 0.28 mmol, 25 equiv). The reaction mixture is stirred for 18 h. The solvents are removed in vacuo and the residue is purified through flash column chromatography (CHCl.sub.3/MeOH 100/0.fwdarw.90/10) to yield yellow oil (25.3 mg, 6.9 μmol, 76%).

(41) [a].sub.D.sup.20=+46.0 (c=1.00 in CHCl.sub.3); ν.sub.max (neat) 3344, 2926, 2864, 2108, 1683, 1497, 1454, 1363, 1093, 1071, 1028, 863 cm.sup.−1; .sup.1H NMR (600 MHz, CDCl.sub.3) δ 7.38-6.79 (m, 105H), 6.23 (s, 1H, NHCOCH.sub.3), 5.87 (d, J=3.4 Hz, 1H), 5.15 (s, 1H), 5.00-3.30 (m, 90H), 3.24 (d, J=10.3 Hz, 1H), 3.11 (d, J=4.6 Hz, 1H), 3.03 (d, J=8.0 Hz, 1H), 2.80 (d, J=10.2 Hz, 1H), 2.69 (q, J=7.2 Hz, 12H, NCH.sub.2CH.sub.3), 2.28 (t, J=7.4 Hz, 2H, BnS—CH.sub.2), 1.86 (s, 1H, NHCOCH.sub.3), 1.59-1.36 (m, 4H, —S—CH.sub.2—CH.sub.2—CH.sub.2—CH.sub.2—CH.sub.2—CH.sub.2—O), 1.26-1.11 (m, 4H, —S—CH.sub.2—CH.sub.2—CH.sub.2—CH.sub.2—CH.sub.2—CH.sub.2—O), 1.02 (t, J=7.3 Hz, 18H, NCH.sub.2CH.sub.3); .sup.13C NMR (151 MHz, CDCl.sub.3) δ 169.72 (NHCOCH.sub.3), 155.80 (O—CO—NH), 139.25, 138.46, 138.30, 138.23, 137.98, 137.70, 137.48, 137.27, 137.00, 136.33, 128.69, 128.25, 128.19, 128.13, 127.85, 127.79, 127.62, 127.27, 127.13, 127.07, 126.80, 126.74, 126.54, 126.26, 125.68, 101.37, 100.31, 99.20, 98.96, 98.52, 94.76, 81.97, 81.82, 81.78, 81.51, 80.99, 80.93, 80.86, 80.84, 80.52, 80.17, 79.87, 79.48, 79.22, 78.90, 78.51, 77.79, 77.30, 75.92, 75.62, 74.89, 74.68, 74.57, 74.19, 73.94, 73.35, 72.99, 72.41, 72.11, 71.48, 71.20, 71.01, 70.46, 69.50, 69.23, 68.93, 67.83, 67.04, 66.60, 66.06, 65.93, 65.65, 64.95, 64.68, 64.19, 64.01, 63.31, 63.23, 62.40, 62.26, 52.47, 51.52, 45.62, 44.66, 43.74, 36.52, 35.62, 34.73, 31.66, 31.10, 30.71, 30.29 (BnS—CH.sub.2), 29.89, 29.82, 29.40, 29.06, 28.55, 28.23, 28.08, 27.72, 27.25, 26.36, 25.59, 24.80, 23.96, 23.12, 22.28 (NHCOCH.sub.3), 9.11, 8.26, 7.41, 6.56; .sup.31P NMR (243 MHz, CDCl.sub.3) δ 0.00, −1.32; m/z (ESI) Found: [M−2H].sup.2−, 1735.2054; C.sub.200H.sub.219N.sub.5O.sub.43P.sub.2S requires [M−2H].sup.2−, 1735.2077.

Example 10: 6-O-(aminoethyl phosphono)-α-D-mannopyranosyl-(1→2)-α-D-mannopyranosyl-(1→6)-4-O-(α-D-glucopyranosyl-(1→4)-2-deoxy-2-acetamido-β-D-galactopyranosyl)-α-D-manno-pyranosyl-(1→4)-2-amino-2-deoxy-α-D-glucopyranosyl-(1→6)-1-O-(thiohexyl phosphono)-D-myo-inositol (Compound 4)

(42) ##STR00020##

(43) Bistriethylammonium 2,3,4-Tri-O-benzyl-6-O-(2-(N-benzyloxycarbonyl)aminoethyl phosphono)-α-D-mannopyranosyl-(1.fwdarw.2)-3,4,6-tri-O-benzyl-α-D-mannopyranosyl-(1.fwdarw.6)-3-O-benzyl-4-O-(2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl-(1.fwdarw.4)-3,6-di-O-benzyl-2-deoxy-2-acetamido-β-D-galactopyranosyl)-α-D-manno-pyranosyl-(1.fwdarw.4)-2-azido-3,6-di-O-benzyl-2-deoxy-α-D-glucopyranosyl-(1.fwdarw.6)-1-O-(6-(S-benzypthiohexyl phosphono)-2,3,4,5-tetra-O-benzyl-D-myo-inositol (compound 14) (20 mg, 5.4 μmol, 1 equiv) was dissolved in dry THF (3 mL) and dry tert-BuOH (2 drops). The solution is cooled down to −78° C. and approximately 10 mL ammonia is condensed in the flask. Afterwards small pieces of sodium are added till a dark blue colour is established. Then the solution is stirred for 35 min at this temperature. The reaction is quenched with 2 mL dry MeOH and the ammonia is blown off of using a stream of nitrogen. Solvents are afterwards evaporated and the residue is dissolved in 5 mL water. The pH of the solution is adjusted with concentrated acetic acid to 7. Water is removed by freeze drying and the residue is purified using a small G25 column (1 cm×20 cm) to yield a white solid (4.5 mg, 3.0 μmol, 55%).

(44) ν.sub.max (neat) 3350, 2918, 1646, 1025 cm.sup.−1; .sup.1H NMR (400 MHz, D.sub.2O) δ 5.59 (d, J=3.6 Hz, 1H, GlcNH.sub.2-1), 5.27 (s, 1H), 5.23 (s, 1H), 5.07 (s, 1H), 4.99 (d, J=3.8 Hz, 1H, Glc-1), 4.60 (d, J=8.2 Hz, 1H, GalNAc-1), 4.31-3.67 (m, 39H), 3.60 (dd, J=10.1, 3.6 Hz, 2H), 3.55-3.39 (m, 3H), 3.35 (t, 2H), 2.83 (t, J=7.2 Hz, 1H), 2.61 (t, J=7.1 Hz, 1H), 2.14 (s, 3H), 1.82-1.58 (m, 4H), 1.54-1.38 (m, 4H); .sup.13C NMR (151 MHz, D.sub.2O) δ 177.28 (amide), 105.01, 104.76 (GalNAc-1), 103.91, 102.82 (Glc-1), 101.11, 99.97 (GlcNH.sub.2-1), 81.64, 79.91, 79.30, 79.25, 79.15, 78.84, 78.04, 75.70, 75.69, 75.39, 75.36, 75.29, 74.84, 74.61, 74.55, 74.40, 74.32, 74.08, 73.94, 73.62, 73.16, 72.86, 72.83, 72.66, 72.58, 72.55, 72.50, 72.27, 72.18, 72.12, 72.06, 71.82, 71.62, 69.60, 69.22, 68.87, 67.29, 64.51, 64.48, 63.77, 62.98, 62.82, 62.52, 56.58, 55.08, 42.72, 42.67, 40.75, 35.52, 32.28, 30.88, 29.80, 29.67, 27.30, 27.18, 26.99, 26.52, 26.29, 25.84, 24.96; .sup.31P NMR (162 MHz, D.sub.2O) δ 0.40, 0.22; m/z (ESI) Found: [M-H].sup.−, 1510.4612; C.sub.52H.sub.93N.sub.3O.sub.41P.sub.2S requires [M-H].sup.−, 1510.4564.

(45) Part A2: Preparation of Sulfone Linked Thiol Functionalized GPI

(46) Formation of the mesilate ester with mesityl chloride and triethylamine followed by a S.sub.N2 reaction leads to bromide 19a that is substituted by the thiol linker under inversion of stereochemistry to generate 19. Oxidation with hydrogen peroxide yields the sulfone 20, which is deprotected under acidic conditions. Introduction of a protected phosphoethanolamine and cleavage of the levulinic ester using hydrazine produces oligosaccharide 21. Final hydrogenolysis yields glycan 22 ready for conjugation. The reaction scheme is shown under FIG. 2.

(47) B Experimental Data for Vaccination

Example 11: Conjugation to a Carrier

(48) Vaccines based on polysaccharides are characterized by a T-cell independent immune response without inducing an immunological memory. Immunogenicity of polysaccharide vaccines in infants, elderly and immunocompromised patients are weak. Conjugation of carbohydrates to a carrier, such as a carrier protein or a glycosphingolipid with immnunomodulatory properties, creates a T-cell dependent immune response against the carbohydrate. As carrier protein, the nontoxic diphtheria toxoid variant CRM.sub.197 was used, since it has been approved as a constituent of licensed vaccines.

Example 11a: Conjugation to a Maleimide-Modified Protein (FIG. 6)

(49) 1 mg (17 nmol) CRM.sub.197 was dissolved in 500 μL PBS to yield a 40 μM CRM.sub.197 solution. 3 mg Sulfo-GMBS (Pierce) was dissolved in 500 μL PBS (1.6 mM, 40 equiv.) and added to the protein solution. The solution was incubated for 2 h at room temperature, before it was concentrated and washed with water (4×500 μL) in an Amicon Ultra-0.5 mL centrifugal filter (Millipore™). Afterwards 250 μg (170 nmol) of GPI 3 was incubated with an equimolar amount of TCEP for 1 h in 500 μL PBS. The GPI solution was added to the concentrated maleimide-modified CRM.sub.197 and the solution was incubated for 3 h at room temperature. The conjugate was purified using a G25 column (10 mm×140 mm, eluent 5% EtOH in water) and the fractions containing the protein were identified using Bradford solution. The fractions containing the conjugate were pooled and the protein concentration was determined by BCA Protein Assay (Pierce). Finally the solution was lyophilized to yield the conjugate as a white solid. Purity and loading were determined via MALDI mass analysis.

Example 11b: In Batch Conjugation to an Olefin-Modified Protein at 254 nm

(50) Compound of general formula (I) (10 equiv.) and olefin-modified CRM.sub.197 (1 equiv., p. Angew. Chem. 2007, 119, 5319) were dissolved in a quartz glass reaction vessel under argon atmosphere in degassed PBS at pH=7.4. The solution was stirred for 6 h under irradiation with light emitted by a low pressure mercury lamp (λ=254, 77 W). Afterwards the solution was frozen dried and the crude material was purified using size exclusion chromatography (Sephadex-G25, 5% EtOH in water, 10 mm×150 mm) to yield the conjugates of the compound of general formula (I) covalently linked to the olefin modified CRM.sub.197, as white solids.

Example 11c: In Flow Conjugation to an Olefin-Modified Protein at 254 nm

(51) By using a photochemical flow reactor (Chem. Eur. J. 2013, 19, 3090) that was fitted with a loop of Teflon AF2400 tubing (566 μL), a solution of compound of general formula (I) (10 equiv.) in water (300 μL) was reacted with olefin-modified CRM.sub.197 (1 equiv., Angew. Chem. 2007, 119, 5319) in water (300 μL) and AcOH (8 μL; residence time: 10 min, flow rate: 28.3 μL/min.sup.−1 per syringe). The reactor output was lyophilized and the crude material was purified using size exclusion chromatography (Sephadex-G25, 5% EtOH in water, 10 mm×150 mm) to yield the conjugates of the compounds of general formula (I) covalently linked to the olefin modified CRM.sub.197 as white solid.

Example 11d: In Flow Conjugation to an Olefin-Modified Protein at 366 nm

(52) By using a photochemical flow reactor (Chem. Eur. J. 2013, 19, 3090) that was fitted with a loop of Teflon AF2400 tubing (566 μL), a solution of compound of general formula (I) (10 equiv.) in water (300 μL) was reacted with olefin modified CRM.sub.197 (1 equiv., Angew. Chem. 2007, 119, 5319) in water (300 μL) and AcOH (8 μL; residence time: 30 min, flow rate: 9.4 μL/min.sup.−1 per syringe). The reactor output was lyophilized and the crude material was purified using size exclusion chromatography (Sephadex-G25, 5% EtOH in water, 10 mm×150 mm) to yield the conjugate of the compound of general formula (I) covalently linked to the olefin modified CRM.sub.197 as white solid.

Example 11e: Conjugation to a α-Iodoacetamide-Modified Protein

(53) Conjugation of the compound 4 to CRM.sub.197 protein is performed as described in FIG. 8. CRM.sub.197 (1 mg, 0.017 μmol) was dissolved in sterile filtered double-distilled water (1 mL) and transferred to an Amicon® Ultra-4 centrifugal filter unit (10 kDa cut-off). To wash away the additive sucrose the solution was concentrated to 200 μL, sterile filtered double-distilled water (800 μL) was added and the solution was concentrated again to 200 μL volume. Phosphate buffer (50 mM NaH.sub.2PO.sub.4, pH 8.5, 800 μL) was added to the solution, which was transferred to an eppendorf tube. Sulfo-SIAB (0.9 mg, 1.7 μmol, Thermo Scientific) was added to the solution, which was agitated for 1 h under the exclusion of light. To wash away unreacted linker the solution was concentrated to 200 μL. Sterile filtered double-distilled water (800 μL) was added and the solution was concentrated again to 200 μL volume. This step was repeated one time. Afterwards, PBS sodium phosphate (pH 8.5, 500 μL) was added to the solution, which was transferred to an eppendorf tube. Compound 4 (250 μg, 0.165 μmol; in 250 μL double-distilled water) that was already incubated for 1 h with an equimolar amount of TCEP.HCl (tris(2-carboxyethyl)phosphine hydrochlorid, Thermo Scientific) was added to the solution. The reaction mixture was agitated for 3 h under the exclusion of light, before a cysteine solution (30 μL, 310 mM) was added to quench unreacted iodoacetamine groups. The conjugate was concentrated again to 200 μL volume. Sterile filtered double-distilled water (800 μL) was added and the solution was concentrated again to 200 μL volume. This step was repeated one time. Sterile filtered double-distilled water (800 μL) was added to the conjugate solution, which was divided in four aliquots of 250 μL each and lyophilized. The white powder was stored at −25° C. before use. Maldi-TOF analysis shows the formation of the target conjugate and that on average three compounds 4 were covalently linked to one carrier protein (see FIG. 9).

Example 11f: In Flow Conjugation of the Compound 4 to a Vinyl-Modified Glycosphingolipid with Immunomodulatory Properties

(54) By using a photochemical flow reactor (Chem. Eur. J. 2013, 19, 3090) that was fitted with a loop of Teflon AF2400 tubing (566 μL), a solution of compound 4 (1.5 equiv.) in water (300 μL) was reacted with pentenyl modified (2S,3S,4R)-1-(α-D-galactopyranosyl)-2-hexacosanoylaminooctadecane-3,4-diol (1 equiv.) in water (300 μL) and AcOH (8 μL; residence time: 10 min, flow rate: 28.3 μL/min.sup.−1 per syringe) (see FIG. 10). The reactor output was lyophilized and the crude material was purified using size exclusion chromatography (Sephadex-G25, 5% EtOH in water, 10 mm×150 mm) to yield the conjugate of compound 4 covalently linked to the pentenyl modified (2S,3S,4R)-1-(α-D-galactopyranosyl)-2-hexacosanoylaminooctadecane-3,4-diol as white solid.

Example 12: Immunizations with the Conjugate Consisting of Compound 3 Covalently Linked to CRM197 Protein

(55) Three female BALB/c mice were immunized s.c. with 35 μg conjugate, prepared as described in example 11a, in Freund's complete adjuvant. All mice were boosted two times with 35 μg conjugate in Freund's incomplete adjuvant in two-week intervals. After the second immunization, serum was collected and the antibody titer (total IgG) was determined by microarray six weeks after the first immunization. The results are shown in FIG. 3.

Example 13: Detection of T. gondii GPIs by Indirect Immunofluorescence

(56) Extracellular tachyzoites collected from cell culture supernatants were fixed with 4% (w/v) paraformaldehyde in PBS for 30 min. Cells were washed three times with PBS, and incubated for 1 h with mice sera raised against compounds 3 or 4 diluted to 100 in PBS, 10% BSA. Cells were washed three times with PBS before incubated for 1 h with secondary FITC-conjugated anti-mouse immunoglobulin antibody (DakoCytomation, Glostrup) containing 10% BSA and washed finally three times with PBS. After three final washes with PBS, aliquot were spotted on microscope slides followed by a glass cover slides, mounted in Fluoroprep (Dako) and recorded by using a 100× Plan-NeoFluar oil objective lens with NA 1.30 using an Axiophot microscop (Zeiss). The results are shown in FIG. 4.

Example 14: Immunizations with the Conjugate Consisting of Compound 4 Covalently Linked to CRM197 Protein

(57) To evaluate the immunogenic properties of the conjugate consisting of compound 4 covalently linked to CRM.sub.197 protein, obtained as described at example 11e, BALB/c mice were immunized and boosted two times with 35 μg conjugate (in each case) in Freund's incomplete adjuvant in two-week intervals. The conjugate proved immunogenic in all mice and immunoglobulin (Ig) class-switching and affinity maturation were detected by carbohydrate microarray analysis (see FIG. 11A). IgG antibodies against compound 4 were detected up to a dilution of 1:1000 in sera of all mice six weeks after the first immunization. The nature of the IgG response was further evaluated, demonstrating that antibodies raised against compound 4 mainly consisted in IgG.sub.1 and IgG.sub.2a subclasses, while IgG.sub.3 was almost indetectable (see FIG. 11B), which is in agreement with previous results (Infect. Immun. 1999, 67, 4862). The high abundance of IgG.sub.2a, which exhibits strong antibody-dependent cellular and complement-dependent cytotoxicity, suggests that the immune response to the conjugate of compound 4 to CRM.sub.197 can induce phagocytosis or lysis of the parasite in vivo, assuming that the antibodies recognize the natural antigen on T. gondii cells.

Example 15: Specificity and Epitope Recognition of the Antibody Response Against Compound 4

(58) To address the specificity and epitope recognition of the antibody response, carbohydrate microarray analysis with substructures of compound 4 was employed (see FIG. 12). The immune response to all animals was highly specific towards compound 4, as antibodies did not recognize any of the substructures of compound 4 (see FIG. 12) at a dilution of 1:1000. This indicates a possible conformational change induced by the α-GcNH.sub.2-(1.fwdarw.6)-myo-Ino moiety that affects the whole glycan 4, since none of the substructures contains this element. Therefore, the structural conformation of compound 4 likely differs from the analyzed substructures, which could explain the preference of the polyclonal antibodies. Another explanation for this specificity might be that the raised antibody recognizes multiple epitopes on compound 4. Hence the avidity of IgGs is significantly lower when one or more structural features are not present.

Example 16: Recognition of the Natural GPI Antigen Displayed on the T. gondii Parasite by the Antibodies Raised Against Compound 4

(59) To confirm that the antibodies raised against compound 4 recognize the natural GPI antigen displayed on the parasite, T. gondii tachyzoites were incubated with serum of immunized mice and analyzed with immunofluoresecence (IF) confocal microscopy (see FIG. 13). The antibodies bound to the surface of the parasite and showed preferential localization of the GPI containing the additional α-glucose in the side chain at the apical end of the cell. In contrast, antibodies binding to the parasite in sera of mice before immunization could not be detected. These results indicate that the GPI containing the additional α-glucose in the side chain potentially plays a role in the formation or function of the apical complex, which is essential for invasion of host cells and plays a critical role during replication of T. gondii. Tachyzoites secrete factors for attachment, invasion and formation of the parasitophorous vacuole, which is surrounding and protecting the parasite inside the host cell from endocytosis, in a regulated fashion from the apical region. Blocking the site of attachment with opsonizing antibodies directed against the GPI structure containing the α-Glc in the side chain and clustering of this antigen could disturb the organization of the apical membrane leading to inhibition of the cell invasion. This dual mechanism of action has great potential to induce sterile immunity against T. gondii