Antibodies for prevention and treatment of diseases caused by clostridium difficile
09815889 · 2017-11-14
Assignee
Inventors
- Peter H. Seeberger (Kleinmachnow, DE)
- Christopher E. Martin (Berlin, DE)
- Felix Broecker (Berlin, DE)
- Chakkumkal Anish (Berlin, DE)
Cpc classification
A61K31/713
HUMAN NECESSITIES
A61K31/715
HUMAN NECESSITIES
C07K16/44
CHEMISTRY; METALLURGY
International classification
A61K31/715
HUMAN NECESSITIES
C07K16/44
CHEMISTRY; METALLURGY
Abstract
The present invention relates to an antibody having specificity for an immunogenic determinant consisting of the pentasaccharide repeating unit of the Clostridium difficile glycopolymer PS-I: α-
Claims
1. An antibody having specificity for an immunogenic determinant comprising the pentasaccharide α-L-Rhap-(1.fwdarw.3)-β-D-Glcp-(1.fwdarw.4)-[α-L-Rhap-(1.fwdarw.3)]-α-D-Glcp-(1.fwdarw.2)-α-D-Glcp or a fragment of the pentasaccharide wherein the antibody is the monoclonal antibody 2C5 (accession number DSM ACC3282), 10A1 (accession number DSM ACC3283), or 10D6 (accession number DSM ACC3284).
2. A vaccine composition comprising at least one antibody according to claim 1 and a pharmaceutically acceptable carrier.
3. A method for treatment or prevention of a disease caused by the pathogen Clostridium difficile, which comprises administering to a subject the antibody according to claim 1.
4. A method for treatment or prevention of a disease caused by the pathogen Clostridium difficile, which comprises administering to a subject the vaccine composition according to claim 2.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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EXAMPLE 1
Preparation and Characterization of a Pentasaccharide Based on the Repeating Unit of C. difficile Polysaccharide PS-I
(25) The pentasaccharide was designed to provide, by means of a linker group, a primary amine at the reducing terminus to facilitate conjugation to a protein carrier and attachment to microarrays and other surfaces. In the following synthesis, the linker comprises the (CH.sub.2).sub.5NH.sub.2 group and the overall synthesis was performed according to scheme 5 or 8 above as indicated.
General Experimental
(26) Commercial grade reagents and solvents were used without further purification except as indicated below. All batch reactions conducted under an Ar atmosphere. .sup.1H-NMR and .sup.13C-NMR spectra were measured with a Varian 400-MR or Varian 600 spectrometer. The proton signal of residual, non-deuterated solvent (δ 7.26 ppm for CHCl.sub.3; δ 4.79 ppm for H.sub.2O, 2.84 ppm for acetone) was used as an internal reference for .sup.1H spectra. For .sup.13C spectra, the chemical shifts are reported relative to the respective solvent (δ 77.16 ppm for CDCl.sub.3, δ 29.84 ppm for acetone). For .sup.13C spectra in D.sub.2O, MeOH (δ 49.50 ppm) was added as internal standard. Coupling constants are reported in Hertz (Hz). The following abbreviations are used to indicate the multiplicities: s, singlet; d, doublet; t, triplet; m multiplet. Infrared (IR) spectra were recorded as thin films on a Perkin Elmer Spectrum 100 FTIR spectrophotometer. Optical rotations (OR) were measured with a Schmidt & Haensch UniPol L 1000 at 589 nm and a concentration (c) expressed in g/100 mL. High-resolution mass spectra (HRMS) were recorded with an Agilent 6210 ESI-TOF mass spectrometer at the Freie Universität Berlin, Mass Spectrometry Core Facility. MALDI-TOF spectra were recorded on a Bruker Daltonics Autoflex Speed. Synthetic carbohydrates were measured using a 2,4,6-trihydroxyacetophenone (THAP) matrix, proteins and glycoconjugates were measured using 2,4-dihydroxyacetophenone (DHAP) as matrix.
(27) Analytical thin layer chromatography (TLC) was performed on Kieselgel 60 F254 glass plates precoated with a 0.25 mm thickness of silica gel. The TLC plates were visualized with UV light and by staining with Hanessian solution (ceric sulfate and ammonium molybdate in aqueous sulfuric acid) or a 1:1 mixture of H.sub.2SO.sub.4 (2N) and resorcine monomethylether (0.2%) in ethanol. Column chromatography was performed using Kieselgel 60 (230-400 mesh). SEC-HPLC analyses were performed on a TSKgel-G4000SWXL column connected to an Agilent 1200 HPLC system equipped with a PDA detector. Elution buffer was constituted by 100 mM sodium phosphate pH 7.2, 100 mM NaCl flow rate was 0.4 mL/min. SDS PAGE gels were run with 10% SDS PAGE gel in reducing conditions at 130 V and 50 mA, molecular weight marker (Invitrogen bench marker) was used.
Synthesis of Pentasaccharide 1 and Intermediates According to Schemes 1-5
Ethyl-3,4,6-tri-O-benzyl-2-O-(2-naphthalenylmethyl)-1-thio-D-glucopyranoside (7)
(28) To a solution of 6 (284 mg, 0.57 mmol) in anhydrous DMF (1 mL), NaH (20.7 mg, 0.86 mmol) followed by NAP-Br (228 mg, 1.03 mmol) were added at 0° C. The mixture was warmed to room temperature over 1 h, cooled to 0° C. and quenched by the addition of MeOH (0.1 mL). Et.sub.2O was added and the organic layer washed with 0.01 M HCl solution and with saturated aqueous NaHCO.sub.3 solution. The phases were separated and the organic layer was dried over MgSO.sub.4 and concentrated. Column chromatography (hexanes/ethyl acetate) afforded 7 (335 mg, 0.53 mmol, 92%) in a mixture of α/β-anomers as a white solid. Analytical data is reported for the β-anomer. [α].sub.D.sup.20+26.1° (c=5.3, CHCl.sub.3), IR ν.sub.max (film) 3061, 3030, 2864, 1949, 1808, 1603, 1497, 1453, 1360, 1065 cm.sup.−1; .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 7.82-7.69 (4H, m, Ar—H), 7.52-7.09 (18H, m, Ar—H), 5.08-5.02 (1H, m, —CH.sub.2—Ar), 4.93-4.77 (4H, m, —CH.sub.2—Ar), 4.60-4.50 (3H, m, —CH.sub.2—Ar), 4.47 (1H, d, J 9.7, 1-H), 3.80-3.54 (4H, m), 3.52-3.41 (2H, m), 2.84-2.66 (2H, m, S—CH.sub.2—), 1.31 (3H, t, J 7.3, CH.sub.3); .sup.13C-NMR (100 MHz, CDCl.sub.3) δ 138.7, 138.4, 138.2, 135.6, 133.4, 133.2, 128.56, 128.55, 128.5, 128.2, 128.1, 127.92, 127.87, 127.84, 127.80, 127.77, 127.7, 127.2, 126.5, 126.1, 126.0, 86.8, 85.2 (C-1), 82.0, 79.3, 78.2, 75.9, 75.7, 75.2, 73.6, 69.3, 25.2, 15.3; HRMS (ESI): Calcd for C.sub.40H.sub.42O.sub.5S [M+Na].sup.+ 657.2651. found 657.2651.
N-(Benzyl)benzyloxycarbonyl-5-amino-pentanyl-3,4,6-tri-O-benzyl-β-D-glucopyranoside (2)
(29) Thioglucoside 7 (335 mg, 0.53 mmol) and HO(CH.sub.2).sub.5NBnCbz (518 mg, 1.58 mmol) were coevaporated with toluene (3×10 ml), dried in vacuo, then the compounds were dissolved in a solution of anhydrous toluene:dioxane=2:1 (4.5 ml). The solution was cooled to −40° C., treated with NIS (131 mg, 0.58 mmol) and TfOH (4.7 μl, 53 μmol) and warmed to −20° C. over 1.5 h. The reaction was quenched with pyridine, diluted with DCM and washed with saturated aqueous Na.sub.2S.sub.2O.sub.3 solution. The organic layer was dried over MgSO.sub.4 and concentrated. Column chromatography on silica gel (hexanes/ethyl acetate) gave a mixture of anomers which was dissolved in DCM (10 ml) and water (1 ml) and treated with DDQ (202 mg, 0.89 mmol) at 0° C. for 2 h. The mixture was diluted with DCM and the organic layer washed with saturated aqueous NaHCO.sub.3 solution, dried over MgSO.sub.4 and concentrated. Column chromatography on silica gel (hexanes/ethyl acetate) afforded 2 (140 mg, 0.184 mmol, 35%) as a colorless oil. [α].sub.D.sup.20=+53.3° (c=5.5), IR ν.sub.max (film) 3458, 3031, 2927, 1952, 1876, 1808, 1454, 1421, 1360, 1229, 1129, 1067 cm.sup.−1; .sup.1H-NMR (400 MHz, acetone-d6) δ 7.48-7.10 (25H, m, Ar—H), 5.15 (2H, bs), 4.99 (1H, d, J 11.4, —CH.sub.2—Bn), 4.84 (1H, d, J 11.1, —CH.sub.2—Bn). 4.79 (1H, d, J 11.4, —CH.sub.2—Bn), 4.75 (1H, bs, 1-H), 4.62-4.49 (5H, m, —CH.sub.2-Bn), 3.84-3.86 (6H, m), 3.62-3.47 (2H, m), 3.40 (1H, m), 3.31-3.18 (2H, m, linker-CH.sub.2—), 1.67-1.50 (4H, m, linker-CH.sub.2—), 1.43-1.29 (2H, m, linker-CH.sub.2—); .sup.13C-NMR (100 MHz, acetone-d6) δ 140.5, 139.8, 139.7, 139.5, 129.3, 129.1, 129.0, 128.9, 128.60, 128.58, 128.43, 128.41, 128.2, 128.0, 99.9 (C-1), 84.3, 78.7, 75.5, 75.4, 74.2, 73.3, 71.5, 70.2, 68.5, 67.4, 24.1; HRMS (ESI): Calcd for C.sub.47H.sub.53NO.sub.8 [M+Na].sup.+ 782.3669. found 782.3633.
(2-Methyl-5-tert-butylphenyl) 2,3,4,6-tetra-O-acetyl-1-thio-β-D-glucopyranoside (9)
(30) 1,2,3,4,6-Penta-O-acetyl-β-
(2-Methyl-5-tert-butylphenyl)-4,6-O-benzylidene-1-thio-β-D-glucopyranoside (10)
(31) Thioglycoside 9 (1.5 g, 2.94 mmol) was dissolved in of methanol (12 mL). Sodium methoxide (58 mg, 1.07 mmol, 0.37 eq) was added and the reaction was stirred over night. After completion, the solution was neutralized with Amberlite IR 120 (H.sup.+) ion exchange resin, filtered and concentrated in vacuo.
(32) The remainder was dried in high vacuum to give (2-Methyl-5-tert-butylphenyl) 1-thio-β-
(2-Methyl-5-tert-butylphenyl)-4,6-O-benzylidene-3-O-tert-butyldimethylsilyl-1-thio-β-D-glucopyranoside (11)
(33) Compound 10 (658 mg, 1.53 mmol) and imidazole (208 mg, 3.06 mmol, 2 eq) were dissolved in anhydrous DMF (880 μL). TBSCl (346 mg, 2.29 mmol, 1.5 eq) was gradually added with stirring. After 4 h, the solvent was evaporated and the resulting oil was dissolved in DCM. The solution was extracted with 1 M HCl and saturated aqueous NaHCO.sub.3 solution, the organic layer was dried over MgSO.sub.4 and the solvent was evaporated in vacuo. The colorless solid was dried in high vacuum and the crude product (820 mg) was purified using flash column chromatography (cyclohexane/ethyl acetate) to afford 11 (573 mg, 1.05 mmol, 69%). [α].sub.D.sup.20=−49.1° (c=1.0, CH.sub.2Cl.sub.2); IR (CH.sub.2Cl.sub.2): 3559, 2957, 2928, 2858, 1631, 1383, 1259, 1110, 1086, 1067, 1009 cm.sup.−1; .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 7.61 (1H, d, J 2.1, Ar—H), 7.51-7.46 (2H, m, Ar—H), 7.39-7.33 (3H, m, Ar—H), 7.26-7.22 (1H, m, Ar—H), 7.15 (1H, d, J 8.0, Ar—H), 5.52 (1H, s, benzylidene-H), 4.65 (1H, d, J 9.8, 1-H), 4.34 (1H, dd, J.sub.1 10.4, J.sub.2 4.4, 6-Ha), 3.84-3.74 (2H, m, 6-Hb, 3-H), 3.54-3.45 (3H, m, 4-H, 5-H, 2-H), 2.42 (3H, s, CH.sub.3), 1.31 (9H, s, tBu), 0.88 (9H, s, tBu), 0.11 (3H, s, CH.sub.3), 0.04 (3H, s, CH.sub.3); .sup.13C-NMR (100 MHz, CDCl.sub.3) δ 149.7, 137.3, 137.0, 131.4, 130.1, 130.1, 129.1, 128.3, 126.3, 125.3 (C-aromatic), 101.8 (C-benzylidene), 89.0 (C-1), 81.2 (C-4), 76.2 (C-3), 74.0 (C-2), 70.8 (C-5), 68.8 (C-6), 31.4 (tBu), 26.0 (tBu), 20.6 (CH.sub.3), −4.2 (CH.sub.3), −4.6 (CH.sub.3); HRMS (ESI): Calcd for C.sub.30H.sub.44O.sub.5SSi [M+Na].sup.+ 567.2571. found 567.2584.
(2-Methyl-5-tert-butylphenyl) 4,6-O-benzylidene-2-O-benzyl-1-thio-β-D-glucopyranoside (12)
(34) To a solution of 11 (2.00 g, 3.67 mmol) in anhydrous DMF (20 ml), NaH (0.21 g, 8.81 mmol) and BnBr (1.31 ml, 11.01 mmol) were added at 0° C. The mixture was warmed to room temperature and stirred over night. Then cooled to 0° C., quenched with MeOH and diluted with Et.sub.2O. The organic layers were washed with H.sub.2O and brine, dried over MgSO.sub.4 and concentrated. Column chromatography on silica gel (hexanes/ethyl acetate) afforded crude (2-methyl-5-tert-butylphenyl) 4,6-O-benzylidene-2-O-benzyl-3-O-tert-butyldimethylsilyl-1-thio-β-
(2-Methyl-5-tert-butylphenyl) 4,6-O-benzylidene-2-O-benzyl-3-O-fluorenylmethoxycarbonyl-1-thio-β-D-glucopyranoside (13)
(35) To a solution of 12 (415 mg, 0.80 mmol) and pyridine (129 μl) in DCM (5 ml), Fmoc-Cl (309 mg, 1.20 mmol) was added and the mixture was stirred over night, diluted with DCM and the organic layers were washed with a 0.01 M HCl solution and saturated aqueous NaHCO.sub.3 solution. The organic layer was dried over MgSO.sub.4 and concentrated. Column chromatography on silica gel (hexanes/ethyl acetate) afforded 13 (561 mg, 0.76 mmol, 95%) as a white solid. [α].sub.D.sup.20=−0.3° (c=5.9, CHCl.sub.3), IR ν.sub.max (film) 3033, 2961, 1955, 1754, 1605, 1451, 1385, 1251, 1077 cm.sup.−1; .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 7.79-7.73 (2H, m, Fmoc-H), 7.65-7.13 (19H, m, Ar—H), 5.55 (1H, s, benzylidene-H), 5.29-5.22 (1H, m), 4.98 (1H, A of AB, J.sub.AB 10.7, —CH.sub.2—Bn), 4.82 (1H, d, J 9.8, H-1), 4.72 (1H, B of AB, J 10.7, —CH.sub.2—Bn), 4.49-4.42 (1H, m), 4.40-4.28 (2H, m), 4.24-4.18 (1H, m), 3.88-3.67 (3H, m), 3.60-3.52 (1H, m), 2.42 (3H, s, CH.sub.3), 1.31 (9H, s, tBu); .sup.13C-NMR (100 MHz, CDCl.sub.3) δ 154.6, 149.8, 143.5, 143.3, 141.4, 137.5, 136.9, 136.6, 130.2, 129.6, 129.2, 128.4, 128.3, 128.2, 128.00, 127.97, 127.30, 127.27, 126.3, 126.2, 125.2, 120.1, 101.6, 88.7 (C-1), 79.5, 79.3, 78.5, 75.7, 70.33, 70.27, 68.8, 46.8, 34.6, 31.4, 20.5; HRMS (ESI): Calcd for C.sub.46H.sub.46O.sub.7S [M+Na].sup.+ 765.2862. found 765.2886.
(2-Methyl-5-tert-butylphenyl)-2,6-di-O-benzyl-3-O-fluorenylmethoxycarbonyl-1-thio-β-D-glucopyranoside (14)
(36) To a solution of 13 (100 mg, 0.14 mmol) in anhydrous DCM (3 ml) freshly activated molecular sieves (4 Å) were added. The mixture was cooled to −78° C., TES (64 μl, 0.40 mmol) and TfOH (41 μl, 0.46 mmol) were added. After stirring for 3 hours at −78° C. the reaction was quenched by the addition of pyridine, diluted with DCM and washed with a saturated aqueous NaHCO.sub.3 solution. The organic phase was then dried over MgSO.sub.4, filtered and concentrated. Column chromatography on silica gel (hexanes/ethyl acetate) afforded 14 (73 mg, 0.10 mmol, 73%). [α].sub.D20=±10.5° (c=4.9, CHCl.sub.3), IR ν.sub.max (film) 3486, 3031, 2959, 1951, 1750, 1604, 1451, 1387, 1254, 1054 cm.sup.−1; .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 7.80-7.74 (2H, m, Fmoc-H), 7.66-7.56 (3H, m, Ar—H), 7.44-7.09 (16H, m, Ar—H), 4.95 (1H, dd, J.sub.1 J.sub.2 9.2, 3-H), 4.92 (1H, d, J 10.7, —CH.sub.2—Bn), 4.69 (1H, d, J 9.8, 1-H), 4.68 (1H, d, J 10.8, —CH.sub.2—Bn), 4.61 (1H, A of AB, J.sub.AB 12.0, —CH.sub.2—Bn), 4.55 (1H, B of AB, J.sub.AB 12.0, —CH.sub.2-Bn), 4.50-4.43 (1H, m, Fmoc-CH.sub.2), 4.40-4.31 (1H, m, Fmoc-CH.sub.2), 4.26-4.20 (1H, m, Fmoc-CH), 3.84 (1H, ddd, J.sub.1 J.sub.2 9.5, J.sub.3 3.6, 4-H), 3.81-3.74 (2H, m, 6-H), 3.61 (1H, dd, J.sub.1 J.sub.2 9.5, 2-H), 3.56-4.49 (1H, m, 5-H), 2.97 (1H, d, J 3.6, 4-OH), 2.40 (1H, s, CH.sub.3), 1.26 (9H, s, tBu); .sup.13C-NMR (100 MHz, CDCl.sub.3) δ 155.7, 149.8, 143.5, 143.4, 141.4, 137.7, 137.6, 136.5, 132.8, 130.1, 129.5, 128.6, 128.4, 128.2 128.04, 127.98, 127.9, 127.3, 125.3, 125.2, 125.0, 120.2, 88.1 (C-1), 83.2 (C-3), 78.5 (C-2), 77.8 (C-5), 75.4, 73.9, 71.0 (C-4), 70.4, 70.3 (C-6), 46.9, 34.6, 31.4, 20.5; HRMS (ESI): Calcd for C.sub.46H.sub.48O.sub.7S [M+Na].sup.+ 767.3018. found 767.3038.
(2-Methyl-5-tert-butylphenyl)-2,6-di-O-benzyl-3-O-fluorenylmethoxycarbonyl-4-O-levulinoyl-1-thio-β-D-glucopyranoside (3)
(37) To a solution of 14 (480 mg, 0.64 mmol) in DCM (8 ml) and pyridine (0.3 ml) Lev.sub.2O (55 mg, 0.26 mmol) was added and stirred for three days. The mixture was diluted with DCM and washed with a 1 M HCl solution and with saturated aqueous NaHCO.sub.3 solution. The organic layers were dried over MgSO.sub.4 and concentrated. Column chromatography on silica gel (hexanes/ethyl acetate) afforded 3 (428 mg, 0.51 mmol, 79%). [α].sub.D.sup.20=+19.2° (c=1.0, CHCl.sub.3), IR ν.sub.max (film) 3065, 2955, 1754, 1719, 1604, 1488, 1452, 1363, 1259, 1152, 1070, 1039 cm.sup.−1; .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 7.80-7.74 (2H, m, Ar—H), 7.68-7.58 (3H, m, Ar—H), 7.44-7.17 (15H, m, Ar—H), 7.15-7.11 (1H, m, Ar—H), 5.20 (1H, dd, J.sub.1 J.sub.2 9.7, 4-H), 5.15-5.07 (1H, m, 3-H), 4.95 (1H, A of AB, J.sub.AB 10.8, —CH.sub.2—Bn), 4.71 (1H, d, J 9.8, 1-H), 4.69 (1H, B of AB, J.sub.AB 10.4, —CH.sub.2-Bn), 4.56-4.41 (3H, m), 4.29-4.20 (2H, m), 3.74-3.55 (4H, m, 2-H, 4-H, 6-H), 2.60-2.52 (2H, m, Lev-CH.sub.2), 2.42 (3H, s, Lev-CH.sub.3), 2.41-2.32 (2H, m, Lev-CH.sub.2), 2.02 (3H, s, SPhCH.sub.3), 1.26 (9H, s, tBu); .sup.13C-NMR (100 MHz, CDCl.sub.3) δ 206.0, 171.6, 154.8, 149.9, 143.7, 143.5, 141.4, 141.3, 138.0, 137.6, 136.6, 132.7, 130.1, 129.5, 128.4, 128.2, 128.1, 128.0, 127.9, 127.7, 127.4, 127.3, 125.5, 125.4, 125.0, 120.1, 88.2, 80.5, 78.9, 77.3, 75.6, 73.7, 70.6, 69.4, 69.2, 46.7, 37.8, 34.6, 31.4, 29.7, 28.0, 20.5; HRMS (ESI): Calcd for C.sub.51H.sub.54O.sub.9S [M+Na].sup.+ 865.3386 found 865.3412.
(2-Methyl-5-tert-butylphenyl) 2-O-benzoyl-4,6-O-benzylidene-3-O-tert-butyldimethylsilyl-1-thio-β-D-glucopyranoside (15)
(38) Thioglycoside 12 (1.00 g, 1.84 mmol) was dissolved under argon in anhydrous pyridine (4 mL). DMAP (67 mg, 0.55 mmol) was added and the solution was cooled to 0° C. BzCl (639 μL, 5.51 mmol) was added dropwise and the solution was heated to 70° C. and stirred for 12 h. After completion (TLC: cyclohexane/ethyl acetate, 9:1), the reaction was quenched with methanol. The suspension was diluted with DCM and extracted with 1 M HCl and H.sub.2O. Column chromatography on silica gel (hexanes/ethyl acetate) afforded 15 (1.05 g, 1.62 mmol, 88%). [α].sub.D.sup.20=+22.9° (c=1.0, CH.sub.2Cl.sub.2); IR (CH.sub.2Cl.sub.2): 2959, 2929, 2858, 1732, 1384, 1266, 1096, 1069 cm.sup.−1; .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 8.08 (2H, dd, J 8.3, Ar—H), 7.56 (1H, d, J 1.8, Ar—H), 7.52-7.43 (5H, m, Ar—H), 7.37 (3H, dd, J.sub.1 5.2, J.sub.2 2.0, Ar—H), 7.20 (1H, dd, J.sub.1 8.0, J.sub.2 2.1, Ar—H), 7.07 (1H, d, J 8.0, Ar—H), 5.58 (1H, s, benzylidene-H), 5.35 (1H, dd, J.sub.1 10.3, J.sub.2 8.6, 2-H), 4.84 (1H, d, J 10.3, 1-H), 4.38 (1H, dd, J.sub.1 10.5, J.sub.2 5.0, 6-Ha), 4.06 (1H, dd, J.sub.1 J.sub.2 8.9, 3-H), 3.88 (1H, dd, J.sub.1 10.3, J.sub.2 5.0, 6-Hb), 3.69 (1H, dd, J.sub.1 J.sub.2 9.1 Hz, 4-H), 3.60-3.52 (1H, m, 5-H), 2.18 (3H, s, CH.sub.3), 1.28 (9H, s, tBu), 0.70 (9H, s, tBu), −0.05 (3H, s, CH.sub.3), −0.14 (3H, s, CH.sub.3); .sup.13C-NMR (100 MHz, CDCl.sub.3) δ 133.1, 129.9, 129.8, 129.4, 129.1, 128.3, 128.1, 126.2, 125.1 (C—Ar), 101.9 (C-benzylidene), 88.1 (C-1), 81.3 (C-4), 74.3 (C-3), 73.6 (C-2), 70.6 (C-5), 68.7 (C-6), 31.3 (tBu), 25.5 (tBu), 20.2 (CH.sub.3), −4.2 (CH.sub.3), −5.0 (CH.sub.3); HRMS (ESI): Calcd for C.sub.37H.sub.48O.sub.6SSi [M+Na].sup.+ 671.2833. found 671.2852.
(2-Methyl-5-tert-butylphenyl) 2-O-benzoyl-4,6-O-benzylidene-1-thio-β-D-glucopyranoside (16)
(39) To a solution of 15 (200 mg, 0.31 mmol) in DMF (1 mL) a solution of TBAF.3H.sub.2O (683 mg, 1.85 mmol) and glacial acetic acid (124 μL, 2.16 mmol) in DMF (1 mL) were added. The mixture was warmed to 35° C. for 9 h, diluted with ether and washed with a 0.01 M HCl solution and saturated aqueous NaHCO.sub.3 solution. The organic layer was dried over MgSO.sub.4 and concentrated. Column chromatography on silica gel (hexanes/ethyl acetate) afforded 16 (150 mg, 0.28 mmol, 91%). [α].sub.D.sup.20=−5.5° (c 0.8, CHCl.sub.3); IR (CHCl.sub.3): 3455, 2963, 2870, 1729, 1268, 1100, 1071 cm.sup.−1; .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 8.11 (2H, d, J 7.4, Ar—H), 7.64-7.33 (9H, m, Ar—H), 7.27-7.20 (1H, m, Ar—H), 7.10 (1H, d, J 8.0, Ar—H), 5.59 (1H, s, benzylidene-H), 5.25 (1H, dd, J.sub.110.1, J.sub.2 8.7, 2-H), 4.88 (1H, d, J 10.1, 1-H), 4.40 (1H, dd, J.sub.1 10.5, J.sub.2 5.0, 6-Ha), 4.09 (1H, dd, J.sub.1 9.0, J.sub.2=8.7, 3-H), 3.87 (1H, dd, J.sub.1 10.4, J.sub.2 5.0, 6-Hb), 3.71 (1H, dd, J.sub.1 9.0, J.sub.2 9.7, 4-H), 3.57 (1H, td, J.sub.1 9.7, J.sub.2 5.0, 5-H), 2.83 (1H, br, 3-OH), 2.23 (3H, s, CH.sub.3), 1.29 (9H, s, tBu); .sup.13C-NMR (100 MHz, CDCl.sub.3) δ 166.1 (C═O benzoyl), 149.7, 137.32, 136.9, 133.6, 131.9, 130.4, 130.2, 129.5, 128.6, 128.5, 126.4, 125.6 (aromatics), 102.1 (C-benzylidene), 87.5 (C-1), 80.9 (C-4), 74.0 (C-3), 73.6 (C-2), 70.5 (C-5), 68.7 (C-6), 31.4 (tBu), 20.4 (CH.sub.3); HRMS (ESI): Calcd for C.sub.31H.sub.34O.sub.6S [M+Na].sup.+ 557.1968. found 557.1975.
(2-Methyl-5-tert-butylphenyl) 2-O-benzoyl-4,6-O-benzylidene-3-O-fluorenylmethoxycarbonyl-1-thio-β-D-glucopyranoside (17)
(40) To a solution of 16 (277 mg, 0.52 mmol) and pyridine (130 μl) in DCM (4 ml), Fmoc-Cl (268 mg, 1.04 mmol) was added and the mixture stirred over night, diluted with DCM and the organic layers were washed with a 0.01 M HCl solution and saturated aqueous NaHCO.sub.3 solution. The organic layer was dried over MgSO.sub.4 and concentrated. Column chromatography on silica gel (hexanes/ethyl acetate) afforded 17 (378 mg, 0.50 mmol, 96%). [α].sub.D.sup.20=+50.2° (c=4.5, CHCl.sub.3), IR ν.sub.max (film) 3066, 2961, 1752, 1732, 1602, 1488, 1450, 1385, 1316, 1268, 1250, 1093 cm.sup.−1; .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 8.06-7.99 (2H, m, Ar—H), 7.73-7.67 (2H, m, Ar—H), 7.61-7.07 (19H, m, Ar—H), 5.60 (1H, s, benzylidene-H), 5.51-5.36 (2H, m, 2-H, 3-H), 4.95 (1H, d, J 9.9, 1-H), 4.46-4.39 (1H, m, 6-H), 4.27-4.16 (2H, m, Fmoc-CH.sub.2), 4.06-4.00 (1H, m, Fmoc-CH), 3.98-3.88 (2H, m, 4-H, 6-H), 3.72-3.63 (1H, m, 5-H) 2.23 (1H, s, CH.sub.3), 1.29 (9H, s, tBu); .sup.13C-NMR (100 MHz, CDCl.sub.3) δ 165.3, 154.6, 149.8, 143.4, 143.2, 141.3, 141.2, 137.4, 136.8, 133.6, 131.7, 130.5, 130.2, 130.1, 129.3, 129.2, 128.5, 128.3, 127.9, 127.27, 127.25, 126.3, 125.8, 125.3, 125.1, 120.00, 119.99, 101.8, 88.0 (C-1), 78.3 (4-H), 77.3 (C-3), 71.4 (C-2), 70.8 (C-5), 70.5, 68.7 (C-6), 46.6, 34.6, 31.7, 31.4, 20.4, 14.3; HRMS (ESI): Calcd for C.sub.46H.sub.44O.sub.8S [M+Na].sup.+ 779.2655. found 779.2649.
Dibutyl-2-O-benzoyl-4,6-O-benzylidene-3-O-fluorenyl-methoxycarbonyl-D-gluco-pyranosidephosphate (4)
(41) Thioglucoside 17 (690 mg, 0.91 mmol) was coevaporated with toluene three times and dried in vacuo, then dissolved in anhydrous DCM (10 ml). Freshly activated molecular sieves (4 Å) and dibutyl hydrogen phosphate (542 μl, 2.73 mmol) were added and the solution cooled to 0° C. NIS (246 mg, 1.09 mmol), followed by TfOH (10 μl, 0.11 mmol) was added and stirred at 0° C. for one hour. The reaction was quenched by the addition of pyridine, diluted with DCM and washed with aqueous Na.sub.2S.sub.2O.sub.3 and saturated aqueous NaHCO.sub.3 solutions. The organic phase was dried over MgSO.sub.4, filtered and concentrated. The crude product was purified by column chromatography on silica gel (hexanes/ethyl acetate) to afford 4 (583 mg, 0.74 mmol, 81%) in a mixture of α/β-anomers (α/β=1:4). NMR data are reported for the β-anomer. [α].sub.D.sup.20=+8.9° (c=3.1, CHCl.sub.3), IR ν.sub.max (film) 3067, 2961, 1755, 1733, 1602, 1451, 1268, 1096, 1026 cm.sup.−1; .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 8.06-7.99 (2H, m, Ar—H), 7.72-7.66 (2H, m, Ar—H), 7.55-7.29 (12H, m, Ar—H), 7.18-7.11 (2H, m, Ar—H), 5.60-5.54 (2H, m, benzylidene-H, 1-H), 5.50 (1H, dd, J.sub.1 J.sub.2 9.4, 2-H), 5.36 (1H, dd, J.sub.1 J.sub.2 9.4, 3-H), 4.49-4.41 (1H, m, 6-H), 4.30-4.18 (2H, m, Fmoc-CH.sub.2), 4.10-4.01 (3H, m, Fmoc-H, phosphate-CH.sub.2), 4.00-3.94 (1H, m, 4-H), 3.90-3.86 (1H, m, 6-H), 3.82-3.67 (3H, m, phosphate-CH.sub.2, 5-H), 1.67-1.60 (2H, m, phosphate-CH.sub.2), 1.42-1.25 (4H, m, phosphate-CH.sub.2), 1.10-1.01 (2H, m, phosphate-CH.sub.2), 0.92 (3H, t, J 7.4, phosphate-CH.sub.3), 0.70 (3H, t, J 7.4, phosphate-CH.sub.3); .sup.13C-NMR (100 MHz, CDCl.sub.3) δ165.1, 154.5, 143.4, 143.1, 141.3, 136.6, 133.8, 130.1, 129.4, 128.6, 128.4, 127.9, 127.2, 126.3, 125.3, 125.2, 120.0, 101.9, 96.91, 96.86, 78.1, 77.5, 77.2, 76.8, 75.8, 72.6, 70.6, 68.4, 68.1, 67.1, 46.6, 32.2, 32.1, 32.0, 31.9, 18.7, 18.4, 13.7, 13.5; δ.sub.P (160 MHz, CDCl.sub.3) −2.95; HRMS (ESI): Calcd for C.sub.43H.sub.47O.sub.12P [M+Na].sup.+ 809.2703. found 809.2690.
4-Methoxyphenyl-2,3-di-O-benzoyl-4-O-benzyl-α-L-rhamnopyranoside (19)
(42) Rhamnoside 18 (500 mg, 1.39 mmol) was dissolved in a solution of DCM (1 ml) and pyridine (1 ml). DMAP (68 mg, 0.56 mmol) was added and the mixture cooled to 0° C., then BzCl (780 mg, 5.56 mmol) was added and the reaction warmed to room temperature over night. The reaction was quenched with MeOH, diluted with DCM and the organic layer was washed with a 0.01 M HCl solution and saturated aqueous NaHCO.sub.3 solution. The organic layer was dried over MgSO.sub.4 and concentrated. Column chromatography on silica gel (hexanes/ethyl acetate) afforded 19 (768 g, 1.35 mmol, 97%). [α].sub.D.sup.20=+17.6° (c=3.1, CHCl.sub.3), IR ν.sub.max (film) 3064, 2934, 1725, 1602, 1506, 1452, 1363, 1273, 1213, 1094, 1027 cm.sup.−1; .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 8.11-8.05 (2H, m, Ar—H), 7.98-7.93 (2H, m, Ar—H), 7.67-7.61 (1H, m, Ar—H), 7.56-7.49 (3H, m, Ar—H), 7.40-7.35 (2H, m, Ar—H), 7.25-7.16 (5H, m, Ar—H), 7.08-7.03 (2H, m, Ar—H), 6.87-6.82 (2H, m, Ar—H), 5.94 (1H, dd, J.sub.1 9.6, J.sub.2 3.4, 3-H), 5.79 (1H, dd, J.sub.1 3.4, J.sub.2 1.9, 2-H), 5.54 (1H, d, J 1.8, 1-H), 4.75 (1H, A of AB, J.sub.AB 10.9, —CH.sub.2—Bn), 4.68 (1H, B of AB, J.sub.AB 10.9, —CH.sub.2-Bn), 4.20-4.11 (1H, m, 5-H), 3.88 (1H, dd, J.sub.1 J.sub.2 9.6, 4-H), 3.78 (3H, s, —CH.sub.3), 1.41 (3H, d, J 6.2, 6-H); .sup.13C-NMR (100 MHz, CDCl.sub.3) δ 165.58, 165.55, 155.21, 150.20, 137.7, 133.6, 133.3, 130.0, 129.9, 129.8, 129.71, 128.69, 128.53, 128.48, 128.2, 128.0, 117.9, 114.7, 96.6 (C-1), 79.1 (C-4), 75.3, 72.3 (C-3), 71.2 (C-2), 68.5 (C-5), 55.8, 18.3 (C-6); HRMS (ESI): Calcd for C.sub.34H.sub.32O.sub.8 [M+Na].sup.+ 591.1995. found 591.1985.
2,3-Di-O-benzoyl-4-O-benzyl-α-L-rhamnopyranoside-N-phenyl-trifluoroacetimidate (5)
(43) CAN (2.17 g, 3.96 mmol) was added to a mixture of 19 (750 mg, 1.32 mmol) in MeCN (12 ml) and H.sub.2O (12 ml) and stirred vigorously for 2 h. H.sub.2O and EtOAc were added, the layers separated, the organic layer washed with H.sub.2O and brine, dried over MgSO.sub.4 and concentrated. Column chromatography on silica gel (hexanes/ethyl acetate) afforded the lactol as an orange solid (548 mg). A solution of the lactol (548 mg) in DCM (10 ml) was cooled to 0° C., CF.sub.3C(NPh)Cl (438 mg, 2.11 mmol) and Cs.sub.2CO.sub.3 (688 mg, 2.11 mmol) were added and the resulting solution was stirred overnight at room temperature, diluted with DCM, filtered through a plug of celite and concentrated. Column chromatography on silica gel (hexanes/ethyl acetate) afforded 5 (619 mg, 0.98 mmol, 74%). [α].sub.D.sup.20+41.2° (c=4.8, CHCl.sub.3), IR ν.sub.max (film) 3065, 2981, 1727, 1600, 1490, 1452, 1270, 1208, 1164, 1091 cm.sup.−1; .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 8.07-8.02 (2H, m, Ar—H), 7.96-7.89 (2H, m, Ar—H), 7.66-7.60 (1H, m, Ar—H), 7.57-7.46 (3H, m, Ar—H), 7.40-7.19 (9H, m, Ar—H), 7.40-7.19 (9H, m, Ar—H), 7.14-7.07 (1H, m, Ar—H), 6.91-6.82 (2H, m, Ar—H), 6.35 (1H, bs, 1-H), 5.84 (1H, s, 2-H), 5.77 (1H, dd, J.sub.1 9.4, J.sub.2 3.3, 3-H), 5.35 (1H, dd, J.sub.1 3.7, J.sub.2 1.9, 1-H), 4.76 (1H, A of AB, J.sub.AB 10.9, —CH.sub.2—Bn), 4.68 (1H, B of AB, J.sub.AB 10.9, —CH.sub.2—Bn), 4.21-4.08 (1H, m, 5-H), 3.87 (1H, dd, J.sub.1 J.sub.2 9.5, 4-H), 1.48 (3H, d, J 6.1, 6-H); .sup.13C-NMR (100 MHz, CDCl.sub.3) δ 165.5, 165.3, 143.4, 137.4, 133.7, 133.4, 130.0, 129.8, 129.7, 129.4, 128.9, 128.7, 128.58, 128.57, 128.3, 128.2, 124.6, 119.6, 94.1 (C-1), 78.5 (C-4), 75.5, 72.0 (C-3), 70.7 (C-3), 69.6 (C-2), 18.4 (C-6); HRMS (ESI): Calcd for C.sub.35H.sub.30F.sub.3NO.sub.7 [M+Na].sup.+ 656.1872. found 656.1852.
N-(Benzyl)benzyloxycarbonyl-5-amino-pentanyl 2,6-di-O-benzyl-3-O-fluorenylmethoxycarbonyl-4-O-levulinoyl-α-D-glucopyranosyl-(1→2)-3,4,6-tri-O-benzyl-α-D-glucopyranoside (20)
(44) Glucoside donor 3 (326 mg, 0.34 mmol) and glucoside acceptor 2 (262 mg, 0.35 mmol) were coevaporated with toluene three times and dried in vacuo. The mixture was dissolved in anhydrous Et.sub.2O (3 ml), NIS (93 mg, 0.41 mmol) was added and cooled to −35° C. TfOH (3.7 μl, 41 μmol) was added and the mixture was stirred and warmed up to −10° C. in one hour. The reaction was quenched by the addition of pyridine, diluted with DCM and washed with aqueous Na.sub.2S.sub.2O.sub.3 and saturated aqueous NaHCO.sub.3 solutions. The phases were separated and the aqueous phase was extracted with DCM. The combined organic phases were dried over MgSO.sub.4, filtered and concentrated. The crude product was purified by column chromatography on silica gel (hexanes/ethyl acetate) to afford 20 (343 mg, 0.24 mmol, 70%). [α].sub.D.sup.20=+64.4° (c=5.9), IR ν.sub.max (film) 3032, 2932, 1755, 1700, 1605, 1497, 1452, 1362, 1259 cm.sup.−1; .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 8.00-6.90 (43H, m, Ar—H), 5.41 (1H, dd, J.sub.1 J.sub.2 9.7), 5.26 (1H, dd, J.sub.1 J.sub.2 9.8), 5.18-5.10 (2H, m), 5.06 (1H, bs, anomeric-H), 5.03-4.96 (2H, m, anomeric-H), 4.88 (1H, app d, J 11.0), 4.82 (1H, app d, J 10.8), 4.68-4.58 (3H, m), 4.52-4.41 (5H, m), 4.39-4.30 (2H, m), 4.26 (1H, app t, J 7.5), 4.14-4.08 (1H, m), 4.07-4.01 (1H, m), 3.82 (1H, dd, J.sub.1 9.9, J.sub.2 3.4), 3.80-3.56 (6H, m), 3.34-3.31 (2H, m), 3.28-3.06 (4H, m), 2.54-2.42 (2H, m), 2.32-2.17 (2H, m), 2.00 (1H, s, Lev-CH.sub.3), 1.65-1.50 (4H, m, linker-CH.sub.2—), 1.30-1.23 (4H, m, linker-CH.sub.2—); .sup.13C-NMR (100 MHz, CDCl.sub.3) δ 206.0, 171.5, 154.9, 143.7, 143.6, 141.40, 141.35, 138.0, 137.8, 128.6, 128.54, 128.53, 128.39, 128.37, 128.2, 128.1, 128.0, 127.94, 127.87, 127.74, 127.66, 127.5, 127.3, 126.3, 125.5, 120.1, 120.0, 95.6 (C-anomeric), 94.0 (C-anomeric), 80.7, 78.2, 77.4, 77.0, 76.8, 76.2, 75.9, 75.4, 73.7, 73.5, 72.4, 70.5, 70.3, 68.8, 68.6, 68.4, 67.3, 46.8, 37.8, 31.4, 29.8, 27.9, 23.7; HRMS (ESI): Calcd for C.sub.87H.sub.91NO.sub.17 [M+Na].sup.+ 1444.6179. found 1444.6128.
N-(Benzyl)benzyloxycarbonyl-5-amino-pentanyl-2,6-di-O-benzyl-3-O-fluorenylmethoxycarbonyl-α-D-glucopyranosyl-(1→2)-3,4,6-tri-O-benzyl-α-D-glucopyranoside (21)
(45) To a solution of 20 (224 mg, 0.16 mmol) in DCM (4.5 ml) hydrazine hydrate (31 μl, 0.63 mmol) dissolved in AcOH (0.4 ml) and pyridine (0.6 ml) was added and the solution stirred for 1 h. The reaction was then quenched by the addition of acetone and concentrated. Column chromatography on silica gel (hexanes/ethyl acetate) afforded 21 (196 mg, 0.15 mmol, 94%). [α].sub.D.sup.20=+57.7° (c=1.7), IR ν.sub.max (film) 3423, 3031, 2926, 1753, 1697, 1605, 1586, 1497, 1452, 1422, 1362, 1255, 1068 cm.sup.−1; .sup.1H-NMR (400 MHz, acetone-d6) δ 7.92-7.84 (2H, m, Ar—H), 7.78-7.64 (2H, m, Ar—H), 7.56-7.14 (35H, m, Ar—H), 5.44-5.37 (2H, m), 5.20-5.10 (3H, m), 5.07 (1H, d, J 10.7), 4.89-4.77 (3H, m), 4.66-4.47 (8H, m), 4.46-4.39 (2H, m), 4.27 (1H, app t, J 6.9), 4-20-4.14 (1H, m), 3.99 (1H, app t, J 9.3), 3.89-3.80 (2H, m), 3.78-3.59 (7H, m), 3.59-3.52 (1H, m), 3.49-3.42 (1H, m), 3.25-3.15 (2H, m), 2.82-2.79 (1H, m), 1.60-1.44 (4H, m, linker-CH.sub.2—), 1.33-1.25 (2H, m, linker-CH.sub.2—); .sup.13C-NMR (100 MHz, acetone-d6) δ 155.9, 144.7, 144.6, 142.2, 142.1, 139.9, 139.8, 139.74, 139.68, 139.5, 139.4, 129.34, 129.26, 129.1, 129.02, 129.00, 128.9, 128.7, 128.62, 128.55, 128.5, 128.4, 128.20, 128.16, 128.14, 128.05, 128.0, 127.9, 126.1, 126.0, 120.88, 120.86, 96.3, 94.2, 81.8, 80.0, 79.2, 78.0, 76.5, 75.5, 73.8, 73.5, 72.1, 71.9, 71.5, 70.2, 70.08, 70.06, 69.5, 68.6, 67.4, 47.6, 27.5, 24.2; HRMS (ESI): Calcd for C.sub.82H.sub.85NO.sub.15 [M+Na].sup.+ 1346.5817. found 1346.5784.
N-(Benzyl)benzyloxycarbonyl-5-amino-pentanyl-2-O-benzoyl-4,6-O-benzylidene-β-D-glucopyranosyl-(1→4)-2,6-di-O-benzyl-α-D-glucopyranosyl-(1→2)-3,4,6-tri-O-benzyl-α-D-glucopyranoside (23)
(46) Phosphate 4 (74 mg, 94 μmol) and 21 (48 mg, 36 μmol) were coevaporated with toluene three times, dried in vacuo and then dissolved in anhydrous DCM (1.0 ml). Freshly activated molecular sieves (4 Å) were added and the mixture cooled to −30° C. TMSOTf (18 μl, 98 μmol) was added and then warmed to −7° C. over 1.5 h. The reaction was quenched with pyridine and concentrated in vacuo. Column chromatography on silica gel (toluene/acetone) afforded crude 22. 20% NEt.sub.3 in DCM (1 ml) was added to crude 22 and stirred for 4 h, the mixture was concentrated in vacuo column chromatography on silica gel (toluene/acetone) afforded 23 (20 mg, 14 μmol, 38%). [α].sub.D.sup.20=+8.1° (c=1.6), IR ν.sub.max (film) 3462, 3032, 2924, 1732, 1699, 1603, 1497, 1453, 1364, 1268, 1093 cm.sup.−1; .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 8.05-7.92 (2H, m, Ar—H), 7.63-7.06 (43H, m, Ar—H), 5.56 (1H, s, benzylidene-H), 5.24 (1H, app t, J 8.5), 5.20-5.11 (2H, m), 5.09-4.98 (2H, m, anomeric-H), 4.88 (1H, app d, J 10.7), 4.79-4.66 (4H, m, anomeric-H), 4.62-4.54 (1H, m), 4.49-4.36 (5H, m), 4.19-4.05 (2H, m), 4.03-3.91 (2H, m), 3.89-3.44 (14H, m), 3.39-3.04 (4H, m), 1.57-1.36 (4H, m, linker-CH.sub.2—), 1.32-1.14 (2H, m, linker-CH.sub.2—); □.sup.13C-NMR (100 MHz, CDCl.sub.3) δ 165.6, 138.51, 138.46, 138.4, 138.1, 136.8, 133.7, 130.1, 129.6, 129.4, 128.7, 128.6, 128.54, 128.52, 128.47, 128.45, 128.4, 127.98, 127.9, 127.84, 127.78, 127.7, 127.4, 126.4, 102.1, 101.7 (C-anomeric), 95.8 (C-anomeric), 94.5 (C-anomeric), 81.1, 80.8, 80.6, 78.2, 77.9, 77.4, 76.1, 75.2, 74.7, 73.6, 73.4, 72.8, 72.1, 71.6, 70.4, 69.5, 68.7, 68.4, 68.2, 67.8, 67.3, 66.4, 29.8, 29.4, 23.6; HRMS (ESI): Calcd for C.sub.87H.sub.93NO.sub.19 [M+Na].sup.+ 1478.6239. found 1478.6136.
N-(Benzyl)benzyloxycarbonyl-5-amino-pentanyl 2,3-di-O-benzoyl-4-O-benzyl-α-L-rhamnopyranosyl-(1→3)-2-O-benzoyl-4,6-O-benzylidene-3-β-D-glucopyranosyl-(1→4)-[2,3-Di-O-benzoyl-4-O-benzyl-α-L-rhamnopyranosyl-(1→3)]-2,6-di-O-benzyl-α-D-glucopyranosyl-(1→2)-3,4,6-tri-O-benzyl-α-D-glucopyranoside (24)
(47) Compounds 5 (26 mg, 41 μmol) and 23 (10 mg, 6.9 μmol) were coevaporated with toluene three times, dried in vacuo and dissolved in anhydrous DCM (1.0 ml). Freshly activated molecular sieves (4 Å) were added and the mixture cooled to −30° C. TMSOTf (10 μl of a solution of 7.4 μl TMSOTf in 93 μl DCM, 4.1 μmol) was added and the reaction was stirred at −30° C. for 1.5 h. The reaction was quenched with pyridine and concentrated in vacuo. Column chromatography on silica gel (toluene/acetone) afforded 24 (14 mg, 5.5 μmol, 81%). [α].sub.D.sup.20=+5.2° (c=0.7), IR ν.sub.max, (film) 3032, 2933, 1728, 1602, 1585, 1496, 1452, 1363, 1263, 1094, 1069 cm.sup.−1; .sup.1H-NMR (400 MHz, CDCl.sub.3) δ8.20-6.90 (75H, m, Ar—H), 5.79-5.67 (3H, m), 5.46 (1H, s, benzylidene-H), 5.33-5.29 (1H, m), 5.28-5.21 (1H, m), 5.17-5.08 (3H, m, anomeric-H), 5.02 (1H, bs, anomeric-H), 4.92-4.78 (4H, m, anomeric-H), 4.74-4.60 (4H, m), 4.59-4.49 (4H, m, anomeric-H), 4.48-4.44 (1H, m), 4.43-4.31 (4H, m), 4.29-4.13 (4H, m, anomeric-H), 4.03-3.88 (3H, m), 3.83-3.45 (13H, m), 3.40-3.02 (7H, m), 1.65 (1H, d, J 6.2, Rha-CH.sub.3), 1.53-1.32 (4H, m, linker-CH.sub.2—), 1.24-1.10 (2H, m, linker-CH.sub.2—), 0.90 (1H, d, J 6.1, Rha-CH.sub.2); □ .sup.13C-NMR (100 MHz, CDCl.sub.3) δ 165.6, 165.48, 165.5, 164.5, 164.2, 138.3, 137.8, 137.6, 133.1, 130.1, 130.0, 129.94, 129.85, 129.7, 129.4, 129.1, 129.0, 128.9, 128.83, 128.76, 128.7, 128.6, 128.51, 128.47, 128.45, 128.42, 128.36, 128.32, 128.29, 128.23, 128.17, 128.04, 128.00, 127.94, 127.88, 127.8, 127.7, 126.5, 126.4, 100.6 (C-anomeric), 100.5 (C-anomeric), 97.9 (C-anomeric), 97.5, 95.8 (C-anomeric), 93.5 (C-anomeric), 80.2, 79.2, 78.1, 77.5, 77.4, 77.2, 76.8, 76.2, 76.1, 76.0, 74.2, 74.0, 73.6, 72.9, 72.1, 71.6, 71.2, 70.9, 68.7, 67.4, 67.2, 50.6, 47.2, 46.2, 29.9, 23.6, 18.4, 17.5; HRMS (ESI): Calcd for C.sub.141H.sub.141NO.sub.31 [M+Na].sup.+ 2366.9385. found 2366.9440.
5-Amino-pentanyl α-L-rhamnopyranosyl-(1→3)-β-D-glucopyranosyl-(1→4)-[α-L-rhamnopyranosyl-(1→3)]-α-D-glucopyranosyl-(1→2)-α-D-glucopyranoside (1)
(48) Fully protected pentasaccharide 24 (10 mg, 4.3 μmol) was dissolved in a solution of NaOMe (0.5 M) in THF/MeOH (1:1, 1 ml) and heated to 50° C. for 12 h. The mixture was neutralized with Amberlite IR 120 (H.sup.+) ion exchange resin, filtered and concentrated. Size exclusion chromatography on Sephadex LH-20 (CHCl.sub.3/MeOH=1:1) afforded the de-benzoylated pentasaccharide (5.6 mg), which was dissolved in a mixture of MeOH (0.9 ml), H.sub.2O (0.1 ml) and AcOH (25 μl). The solution was purged with Argon, 10% Pd/C (10 mg) was added and the solution purged with H.sub.2 for 30 min, then stirred under an H.sub.2 atmosphere for 12 h, filtered and concentrated. Size exclusion chromatography on Sephadex LH-20 (MeOH) afforded 1 (2.3 mg, 2.6 μmol, 61%). NMR data are reported in Table 1, comparison with the data from native PS-I is reported in Table 2. HRMS (MALDI-TOF): Calcd for C.sub.35H.sub.63NO.sub.24 [M+Na].sup.+ 904.3632. found 904.3606.
(49) TABLE-US-00001 TABLE 1 .sup.1H NMR δ (600 MHz, D.sub.2O) and .sup.13C NMR δ (150 MHz, D.sub.2O) of pentasaccharide 1..sup.a α-Glc α-Glc β-Glc α-Rha α-Rha (A) (B) (C) (D) (D′) Linker H-1 5.18 5.09 4.53 5.24 5.14 C-1 96.1 96.8 102.4 101.8 102.0 H-2 3.70 3.73 3.38 4.06 4.06 C-2 72.7 73.4 75.3 71.4 71.2 H-3 3.70 4.03 3.61 3.88 3.81 C-3 76.1 77.0 83.2 71.1 71.2 H-4 3.48 3.86 3.46 3.47 3.47 C-4 70.5 73.8 69.1 73.0 73.0 H-5 3.82 4.05 3.45 4.43 4.03 C-5 72.5 72.3 77.2 69.5 69.8 H-6 3.88/3.78 3.92 3.80/3.96 1.27 1.27 a/b C-6 61.6 60.3 62.2 17.5 17.5 H-1′ 3.79/3.59 a/b C-1′ 68.7 H-2′ 1.70 C-2′ 29.0 H-3′ 1.49 C-3′ 23.5 H-4′ 1.70 C-4′ 27.7 H-5′ 3.01 C-5′ 40.4 .sup.a.sup.1H and .sup.13C NMR resonances were assigned based on HSQC, HMBC, COSY and TOCSY experiments.
(50) TABLE-US-00002 TABLE 2 Comparison of .sup.1H and .sup.13C NMR δ between 1 and the native PS-I repeating unit..sup.a α-Glc α-Glc β-Glc α-Rha α-Rha (A) (B) (C) (D) (D′) H-1 5.18 5.09 4.53 5.24 5.14 5.75 5.13 4.53 5.23 5.17 C-1 96.1 96.8 102.4 101.8 102.0 93.5 98.0 102.4 101.9 101.4 H-2 3.70 3.73 3.38 4.06 4.06 3.68 3.70 3.38 4.07 4.09 C-2 72.7 73.4 75.3 71.4 71.2 77.3 73.6 75.2 71.1 71.2 H-3 3.70 4.03 3.61 3.88 3.81 3.89 4.01 3.62 3.85 3.97 C-3 76.1 77.0 83.2 71.1 71.2 72.1 77.5 83.0 71.0 70.9 H-4 3.48 3.86 3.46 3.47 3.47 3.53 3.86 3.46 3.46 4.07 C-4 70.5 73.8 69.1 73.0 73.0 70.1 73.6 69.1 73.0 78.9 H-5 3.82 4.05 3.45 4.43 4.03 3.91 4.06 3.45 4.44 4.12 C-5 72.5 72.3 77.2 69.5 69.8 73.8 72.4 77.1 69.4 68.6 H-6 3.88/3.78 3.92 3.80/3.96 1.27 1.27 a/b n.d. n.d. 3.80/3.95 1.27 1.33 C-6 61.6 60.3 62.2 17.5 17.5 n.d. n.d. 62.2 17.5 17.8 .sup.adata of native PS-I reported in italic taken from: J. Ganeshapillai et al., Carbohydr. Res., 2008, 343, 703.
Synthesis of Pentasaccharide 1 and Intermediates According to Schemes 6-8
(2-Methyl-5-tert-butylphenyl) 4,6-O-benzylidene-2-O-benzyl-3-O-(4-bromo)benzyl-1-thio-β-D-glucopyranoside (25)
(51) To a solution of 12 (200 mg, 0.38 mmol) in anhydrous DMF (2 ml), NaH (22 mg, 0.92 mmol) was added followed by para-bromobenzyl (PBB) bromide (288 mg, 1.15 mmol) at 0° C. The mixture was warmed to room temperature over 2 h, cooled to 0° C. and quenched by the addition of MeOH. Et.sub.2O was added and the organic layer washed with 0.1 M HCl solution and with saturated aqueous NaHCO.sub.3 solution. The phases were separated and the organic layer was dried over MgSO.sub.4 and concentrated. Column chromatography (cyclohexane/ethyl acetate) afforded 25 (276 mg) along with aromatic impurities and was taken to the next step without further purification.
(2-Methyl-5-tert-butylphenyl) 2,6-di-O-benzyl-3-O-(4-bromo)benzyl-1-thio-β-D-glucopyranoside (26)
(52) To a solution of 25 (140 mg, 0.20 mmol) in anhydrous DCM (4 ml) freshly activated molecular sieves (4 Å) were added. The mixture was cooled to −78° C., TES (97 μl, 0.61 mmol) and TfOH (61 μl, 0.69 mmol) were added. After stirring for 3 hours at −78° C., the reaction was quenched by the addition of saturated aqueous NaHCO.sub.3 solution, diluted with DCM and washed with a saturated aqueous NaHCO.sub.3 solution. The organic phase was then dried over MgSO.sub.4, filtered and concentrated. Column chromatography on silica gel (cyclohexane/ethyl acetate) afforded 26 (81 mg, 0.12 mmol, 58%). .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 7.65-7.60 (m, 1H, ArH), 7.54-7.10 (m, 16H, ArH), 4.98 (d, 1H, J=10.3 Hz, benzyl), 3.65-3.44 (m, 6H, benzyl, 1-H), 3.79-3.70 (m, 3H, 6-H, 4-H), 3.56-3.43 (m, 3H, 2-H, 3-H, 5-H), 2.76 (d, 1H, J=2.2 Hz, 4-OH), 2.40 (s, 3H, CH.sub.3), 1.26 (s, 9H, tBu); .sup.13C-NMR (100 MHz, CDCl.sub.3) δ 149.7, 138.1, 137.72, 137.67, 136.1, 133.3, 131.7, 130.0, 129.6, 128.9, 128.6, 128.5, 128.3, 128.0, 128.0, 124.7, 121.8, 88.2 (C-1), 86.2 (C-2), 80.7 (C-3), 77.5 (C-5), 75.7, 74.7, 73.9, 72.7 (C-4), 70.7 (C-6), 31.4, 20.5; HRMS (ESI): Calcd for C.sub.38H.sub.43BrO5SNa.sup.+ [M+Na].sup.+ 713.1907. found 713.1951.
(2-Methyl-5-tert-butylphenyl) 2,6-di-O-benzyl-3-O-(4-bromo)benzyl-4-O-levulinoyl-1-thio-β-D-glucopyranoside (27)
(53) To a solution of 26 (1.55 g, 2.24 mmol) in DCM (20 ml) at 0° C., DMAP (274 mg, 2.24 mmol), LevOH (1.30 ml, 11.20 mmol) and DCC (2.31 g, 11.20 mmol) were added. The solution was warmed to room temperature and stirred for 16 h. The reaction was diluted with DCM and the organic layers were washed with a 0.1 M HCl solution and saturated aqueous NaHCO.sub.3 solution. The organic layer was dried over MgSO.sub.4 and concentrated. Column chromatography on silica gel (hexanes/ethyl acetate) afforded 27 (1.54 g, 1.95 mmol, 87%). [α].sub.D.sup.20=+6.4° (c=3.4, CHCl.sub.3), IR ν.sub.max (film) 2963, 1744, 1718, 1488, 1361, 1261, 1068, 1038, 1012 cm.sup.−1; .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 7.70-7.05 (m, 17H, Ar—H), 5.11-5.04 (m, 1H, 4-H), 4.97 (app. d, 1H, J=10.4 Hz, benzyl-H), 4.77-4.60 (m, 4H, benzyl-H, 1-H), 4.48 (s, 1H, PBB-H), 3.70-3.54 (m, 5H, 2-H, 3-H, 5-H, 6-H), 2.64-2.55 (m, 2H, Lev-CH.sub.2), 2.40 (s, 3H, S—CH.sub.3), 2.35-2.29 (m, 2H, Lev-CH.sub.2), 2.12 (s, 3H, Lev-CH.sub.3), 1.25 (s, 9H, tBu); .sup.13C-NMR (100 MHz, CDCl.sub.3) δ 206.2 (Lev-carbonyl), 171.7, 149.8, 138.1, 138.0, 137.5, 136.2, 133.2, 131.6, 130.0, 129.6, 128.9, 128.5, 128.4, 128.3, 128.1, 128.0, 127.69, 124.71, 121.6, 88.3 (C-1), 84.1, 81.1, 77.4, 75.8, 74.5, 73.7, 71.3, 69.7, 37.8, 34.6, 31.4, 29.9, 28.0, 20.5; HRMS (MALDI-TOF): Calcd for C.sub.43H.sub.49BrO.sub.7SNa.sup.+ [M+Na].sup.+ 811.2275. found 811.2026.
(2-Methyl-5-tert-butylphenyl) 2-O-benzoyl-4-O-benzyl-3-O-tert-butyldimethylsilyl-1-thio-β-D-glucopyranoside (28)
(54) To a solution of 15 (800 mg, 1.23 mmol) in anhydrous DCM (12 ml) freshly BH.sub.3.THF (1 M in THF, 7.4 ml, 7.4 mmol) and TMSOTf (0.11 ml, 0.62 mmol) were added drop wise at 0° C. The reaction was warmed to room temperature over 2 hours, cooled to 0° C. again and quenched by the drop wise addition of saturated aqueous NaHCO.sub.3 solution. The Emulsion was diluted with DCM and washed with a saturated aqueous NaHCO3 solution. The organic phase was then dried over MgSO4, filtered and concentrated. Crude 28 was taken to the next step.
(2-Methyl-5-tert-butylphenyl) 2-O-benzoyl-4,6-di-O-benzyl-3-O-tert-butyldimethylsilyl-1-thio-β-D-glucopyranoside (29)
(55) To a solution of crude 28 (approx. 1.23 mmol) in THF/DMF (9:1, 10 ml) at 0° C., BnBr (0.18 ml, 1.50 mmol) and NaH (36 mg, 1.50 mmol) were added. The solution was warmed to room temperature over 2 h, then cooled to 0° C. again and further BnBr (0.18 ml, 1.50 mmol) was added. The reaction was warmed to room temperature over 30 min, cooled to 0° C. and quenched by the addition of water. After dilution with Et.sub.2O the phases were separated and the aqueous layer extracted with Et.sub.2O. The organic phase was then dried over MgSO4, filtered and concentrated. Column chromatography on silica gel (hexanes/ethyl acetate) afforded 29 (797 mg, 1.08 mmol, 88%). .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 8.14-7.00 (m, 18H, Ar—H), 5.31 (dd, 1H, J.sub.1=10.1 Hz, J.sub.2=8.9 Hz, 2-H), 4.83 (app. d, 1H, J=11.3 Hz, benzyl-H.sub.a), 4.72 (d, 1H, J=10.2 Hz, 1-H), 4.63 (app. d, 1H, J=11.0 Hz, benzyl-H.sub.b), 4.58 (app. d, 2H, J=3.1 Hz, benzyl-H), 3.95 (app. t, 1H, J=8.7 Hz, 3-H), 3.78-3.51 (m, 4H, 4-H, 5-H, 6-H), 2.15 (s, 3H, S—CH.sub.3), 1.25 (s, 9H, S-tBu), 0.79 (s, 9H, TBS-tBu), 0.00 (s, 3H, TBS-CH.sub.3), −0.16 (s, 3H, TBS-CH.sub.3); .sup.13C-NMR (100 MHz, CDCl.sub.3) δ 165.6, 149.7, 138.2, 136.5, 133.2, 130.5, 130.1, 129.8, 129.2, 128.5, 128.4, 128.0, 127.72, 127.68, 127.6, 124.7, 88.0 (C-1), 79.5 (C-5), 78.9 (C-4), 77.0 (C-3), 75.1, 73.6 (C-2), 73.5, 69.0 (C-6), 31.4, 25.8, 20.3, −3.9, −4.1; HRMS (ESI): Calcd for C.sub.44H.sub.56O.sub.6SSiNa.sup.+ [M+Na].sup.+ 763.3459. found: 763.3500.
N-(Benzyl)benzyloxycarbonyl-5-amino-pentanyl 2,6-di-O-benzyl-3-O-(4-bromo)benzyl-α-D-glucopyranosyl-(1→2)-3,4,6-Tri-O-benzyl-α-D-glucopyranoside (30)
(56) Thioglucoside 27 (323 mg, 0.41 mmol) and glucoside 2 (222 mg, 0.29 mmol) were coevaporated with toluene three times and dried in vacuo. The mixture was dissolved in Ether (4 ml), freshly activated and acid washed molecular sieves (4 Å) and NIS (105 mg, 0.47 mmol) were added and cooled to −40° C. TfOH (4.2 μl, 0.05 mmol) was added and the mixture was stirred and warmed up to −10° C. in one hour. The reaction was quenched by the addition of pyridine, diluted with DCM and washed with aqueous Na.sub.2S.sub.2O.sub.3 and saturated aqueous NaHCO.sub.3 solutions. The phases were separated and the aqueous phase was extracted with DCM. The combined organic phases were dried over MgSO.sub.4, filtered and concentrated. The crude product was purified by column chromatography on silica gel (toluene/acetone) to afford 30 (276 mg, 0.20 mmol, 69%). [α].sub.D.sup.20=+54.1° (c=4.8, CHCl.sub.3), IR ν.sub.max, (film) 3031, 2923, 2864, 1744, 1698, 1497, 1454, 1420, 1360, 1209 cm.sup.−1; .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 7.60-7.02 (m, 39H, Ar—H), 5.24-5.10 (m, 3-H), 5.09-4.93 (m, 3H, 2× anomeric-H), 4.89-4.37 (m, 13H), 4.13-4.00 (m, 2H), 3.99-3.56 (m, 8H), 3.50-3.08 (m, 5H), 2.63-2.47 (m, 2H), 2.25-2.18 (m, 2H), 2.13 (s, 3H, Lev-CH.sub.3), 1.71-1.38 (m, 4H, linker-H), 1.36-1.14 (m, 2H, linker-H); .sup.13C-NMR (100 MHz, CDCl.sub.3) δ 206.3 (Lev-carbonyl), 171.4, 138.7, 138.3, 138.1, 138.1, 137.8, 131.4, 129.6, 128.7, 128.5, 128.4, 128.3, 128.1, 128.03, 127.98, 127.93, 127.88, 127.8, 127.60, 127.57, 127.4, 121.4, 95.5 (C-anomeric), 93.5 (C-anomeric), 80.9, 79.3, 78.8, 78.1, 75.7, 75.3, 74.1, 73.7, 73.5, 72.3, 70.5, 70.2, 68.6, 68.3, 68.1, 67.3, 37.8, 30.0, 29.5, 27.9, 23.7; HRMS (MALDI-TOF): Calcd for C.sub.79H.sub.86BrNO.sub.15Na.sup.+ [M+Na].sup.+ 1390.5073. found 1390.5105.
N-(Benzyl)benzyloxycarbonyl-5-amino-pentanyl 2,6-di-O-benzyl-3-O-(4-bromo)benzyl-α-D-glucopyranosyl-(1→2)-3,4,6-Tri-O-benzyl-α-D-glucopyranoside (31)
(57) To a solution of 30 (300 mg, 0.22 mmol) in DCM (5.0 ml) hydrazine hydrate (32 μl, 0.66 mmol) dissolved in AcOH (0.4 ml) and pyridine (0.6 ml) was added and the solution stirred for 1 h. The reaction was then quenched by the addition of acetone and concentrated. Column chromatography on silica gel (hexanes/ethyl acetate) afforded 31 (117 mg, 0.09 mmol, 96%). [α].sub.D.sup.20+56.5° (c=2.7, CHCl.sub.3), IR ν.sub.max (film) 3453, 2963, 1695, 1454, 1420, 1360, 1259, 1013 cm.sup.−1; .sup.1H-NMR (600 MHz, CDCl.sub.3) δ 7.90-7.00 (39H, m, Ar—H), 5.25-5.13 (m, 2H), 5.10 (bs, 1H, anomeric-H), 5.05 (bs, 1H, anomeric-H), 4.98-4.43 (m, 14H), 4.10-3.53 (m, 13H), 3.45-3.10 (m, 3H), 1.65-1.40 (m, 4H, linker-H), 1.34-1.15 (m, 2H, linker-H); .sup.13C-NMR (150 MHz, CDCl.sub.3) δ 138.7, 138.2, 138.1, 131.6, 129.7, 128.6, 128.49, 128.45, 128.1, 128.0, 127.97, 127.91, 127.85, 127.74, 127.71, 127.3, 121.6, 95.6 (C-anomeric), 93.9 (C-anomeric), 81.4, 81.0, 78.9, 78.1, 77.4, 77.2, 77.0, 75.8, 75.2, 74.4, 73.6, 73.6, 72.1, 71.1, 70.5, 69.3, 68.6, 68.3, 67.3, 50.3, 47.2, 46.2, 43.3, 29.5, 27.7, 23.6; HRMS (MALDI-TOF): Calcd for C.sub.74H.sub.80BrNO.sub.13Na.sup.+ [M+Na].sup.+ 1292.4705. found 1292.4701.
N-(Benzyl)benzyloxycarbonyl-5-amino-pentanyl 2-O-benzoyl-4,6-di-O-benzyl-3-O-tert-butyldimethylsilyl-α-D-glucopyranosyl-(1→4)-2,6-di-O-benzyl-3-O-(4-bromo)benzyl-α-D-glucopyranosyl-(1→2)-3,4,6-tri-O-benzyl-α-D-glucopyranoside (32)
(58) Thioglucoside 29 (233 mg, 0.31 mmol) and disaccharide 31 (266 mg, 0.21 mmol) were coevaporated with toluene three times and dried in vacuo. The mixture was dissolved in DCM (7 ml), freshly activated and acid washed molecular sieves (4 Å) and NIS (80 mg, 0.36 mmol) were added and cooled to −30° C. TfOH (3.2 μl, 0.04 mmol) was added and the mixture was stirred and warmed up to −17° C. in one hour. The reaction was quenched by the addition of pyridine, diluted with DCM and washed with aqueous Na.sub.2S.sub.2O.sub.3 and saturated aqueous NaHCO.sub.3 solutions. The phases were separated and the aqueous phase was extracted with DCM. The combined organic phases were dried over MgSO.sub.4, filtered and concentrated. The crude product was purified by column chromatography on silica gel (toluene/acetone) to afford 32 (354 mg, 0.19 mmol, 92%). [α].sub.D.sup.20=+52.5° (c=2.6, CHCl.sub.3), IR ν.sub.max (film) 3031, 2928, 2859, 1733, 1699, 1603, 1497, 1454, 1421, 1362, 1314, 1265, 1070 cm.sup.−1; .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 7.91-7.05 (m, 54H, Ar—H), 5.21-5.11 (m, 3H), 5.04 (bs, 1H, anomeric-H), 5.01-4.95 (m, 2H, anomeric-H), 4.81 (app. d, 1H, J=11.3 Hz), 4.74-4.35 (m, 17H, anomeric-H), 4.23 (app. d, 1H, J=12.3 Hz), 3.98 (app. t, 1H, J=9.4 Hz), 3.93-3.87 (m, 1H), 3.82 (app. t, 1H, J=9.3 Hz), 3.74-3.66 (m, 4H), 3.64-3.45 (m, 10H), 3.42-3.36 (m, 1H), 3.34-3.06 (m, 4H), 1.56-1.35 (m, 4H), 1.25-1.09 (m, 2H), 0.79 (s, 9H, tBu), 0.02 (s, 3H), −0.19 (s, 3H); .sup.13C-NMR (100 MHz, CDCl.sub.3) δ 164.8, 138.7, 138.7, 138.4, 138.4, 138.4, 138.1, 133.1, 131.1, 130.1, 130.0, 129.6, 128.7, 128.6, 128.49, 128.48, 128.43, 128.41, 128.40, 128.37, 128.35, 128.2, 128.0, 127.93, 127.86, 127.8, 127.7, 127.64, 127.58, 127.5, 127.4, 120.8, 100.3 (C-anomeric), 96.1 (C-anomeric), 59.0 (C-anomeric) 80.5, 80.0, 79.1, 78.6, 77.7, 76.1, 75.5, 75.4, 75.3, 75.2, 74.7, 74.4, 73.8, 73.6, 73.5, 72.3, 70.6, 70.3, 69.1, 68.7, 67.6, 67.2, 50.6, 47.2, 46.3, 29.4, 28.1, 25.8, 23.6, 17.9, −3.86, −3.89; HRMS (MALDI-TOF): Calcd for C.sub.107H.sub.120BrNO.sub.19SiNa.sup.+ [M+Na].sup.+ 1852.7299 found 1852.7375.
N-(Benzyl)benzyloxycarbonyl-5-amino-pentanyl 2-O-benzoyl-4,6-di-O-benzyl-β-D-glucopyranosyl-(1→4)-2,6-di-O-benzyl-α-D-glucopyranosyl-(1→2)-3,4,6-tri-O-benzyl-α-D-glucopyranoside (33)
(59) A solution of 32 (100 mg, 0.06 mmol), (3,4-dimethoxyphenyl)boronic acid (20 mg, 0.11 mmol), TBABr (1.8 mg, 5.5 μmol), K.sub.3PO.sub.4 (35 mg, 0.16 mmol) in EtOH (4 ml) was subjected to three freeze-pump-saw cycles. To this solution Pd(OAc).sub.2 (1.2 mg, 5.5 μmol) was added and stirred for 2 hours. The mixture was diluted with EtOAc and washed with saturated aqueous NaHCO.sub.3 solution. The aqueous phase was back extracted with EtOAc. The combined organic phases were dried over MgSO.sub.4, filtered and concentrated. The crude product was purified by column chromatography on silica gel (toluene/acetone) to afford the Suzuki coupling product (95 mg, 0.05 mmol, 92%) which was dissolved in DCM/H.sub.2O/saturated aqueous NaHCO.sub.3 (100:9:1, 11 ml). To this emulsion DDQ (34 mg, 0.15 mmol) was added, stirred vigorously for 16 hours, diluted with DCM and washed with saturated aqueous NaHCO.sub.3 solutions. The combined organic phases were dried over MgSO.sub.4, filtered and concentrated. The crude product was dissolved in DMF (2.5 ml), and treated with a solution of TBAF.3H.sub.2O (137 mg, 0.43 mmol) and AcOH (29 μl, 0.51 mmol) in DMF (2.5 ml) at 50° C. for three days. After dilution with Et.sub.2O the phases were separated and the organic phase washed with a 0.1 M HCl solution, saturated aqueous NaHCO.sub.3 solution and brine. The organic phase was then dried over MgSO4, filtered and concentrated. The crude product was purified by column chromatography on silica gel (toluene/acetone) to afford 33 (52 mg, 0.03 mmol, 68%). [α].sub.D.sup.20=+38.9° (c=1.5, CHCl.sub.3), IR ν.sub.max (film) 3462, 3031, 2924, 2867, 1729, 1699, 1497, 1454, 1422, 1362, 1315, 1268, 1095, 1069 cm.sup.−1; .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 8.07-7.00 (m, 50H), 5.25-5.05 (m, 3H), 5.03-4.94 (m, 2H, 2× anomeric-H), 4.90 (app. d, J=10.6, 1H), 4.82-4.35 (m, 16H), 4.27 (app. d, J=12.1, 1H), 4.16 (app. dd, J=9.2, 8.8, 1H), 4.06 (app. d, J=12.2, 1H), 3.99 (app. t, J=9.3, 1H), 3.93-3.42 (m, 15H), 3.28 (s, 4H), 1.73-1.36 (m, 4H, linker-H), 1.34-1.08 (m, 2H, linker-H); .sup.13C-NMR (100 MHz, CDCl.sub.3) δ 166.2, 139.0, 138.5, 138.4, 137.9, 137.7, 133.6, 130.1, 129.4, 128.72, 128.65, 128.62, 128.59, 128.57, 128.52, 128.46, 128.45, 128.40, 128.36, 128.30, 128.24, 128.18, 127.97, 127.95, 127.93, 127.88, 127.8, 127.61, 127.57, 127.37, 101.2 (C-anomeric), 95.9 (C-anomeric), 94.8 (C-anomeric), 81.6, 78.4, 78.2, 78.0, 77.5, 77.4, 77.2, 76.8, 76.6, 76.3, 75.0, 74.7, 73.9, 73.6, 73.2, 72.7, 72.2, 70.4, 69.3, 69.1, 68.7, 67.3, 50.4, 47.3, 29.5, 28.1, 23.6; HRMS (MALDI-TOF): Calcd for C.sub.94H.sub.101NO.sub.19Na.sup.+ [M+Na].sup.+ 1570.6860. found 1570.6362.
N-(Benzyl)benzyloxycarbonyl-5-amino-pentanyl 2,3-di-O-benzoyl-4-O-benzyl-α-L-rhamnopyranosyl-(1→3)-2-O-benzoyl-4,6-O-benzyl-β-D-glucopyranosyl-(1→4)-[2,3-di-O-benzoyl-4-O-benzyl-α-L-rhamnopyranosyl-(1→3)]-2,6-di-O-benzyl-α-D-glucopyranosyl-(1→2)-3,4,6-tri-O-benzyl-α-D-glucopyranoside (34)
(60) Rhamnosyl-imidate 5 (72 mg, 140 μmol) and trisaccharide 33 (42 mg, 27 μmol) were coevaporated with toluene three times, dried in vacuo and dissolved in anhydrous DCM (3.0 ml). Freshly activated molecular sieves (4 Å) were added and the mixture cooled to −40° C. TMSOTf (25 μl of a solution of 100 μl TMSOTf in 900 μl DCM, 14 μmol) was added and the reaction was warmed to −20° C. over 1.5 h. The reaction was quenched with TEA and concentrated. Size exclusion chromatography on Sephadex LH-20 (CHCl.sub.3/MeOH 1:1) afforded 34 (58 mg, 24 μmol, 88%). [α].sub.D.sup.20=+49.7° (c=2.2, CHCl.sub.3), IR ν.sub.max (film) 3031, 2927, 2863, 1729, 1700, 1602, 1497, 1453, 1273, 1264, 1095, 1069 cm.sup.−1; .sup.1H-NMR (600 MHz, CDCl.sub.3) δ 8.08-6.98 (m, 80, Ar—H), 5.89 (app. dd, J=9.4, 3.5, 1H), 5.85 (app. dd, J=3.5, 1.7, 1H), 5.66 (app. dd, J=9.4, 3.5, 1H), 5.49-5.42 (m, 1H), 5.39 (app. dd, J=3.5, 1.8, 1H), 5.32-5.25 (m, 1H), 5.16-5.12 (m, 2H), 5.085.05 (m, 1H), 5.04-4.99 (m, 1H), 4.97 (d, J=1.6, 1H), 4.954.25 (m, 20H), 4.24-3.43 (m, 20H), 3.39-3.00 (m, 5H), 1.67 (d, J=6.2, 3H), 1.60-1.32 (m, 4H), 1.32-1.06 (m, 2H), 0.94 (d, J=6.1, 3H); .sup.13C-NMR (150 MHz, CDCl.sub.3) δ 165.43, 165.21, 164.49, 164.12, 139.19, 138.54, 138.37, 138.16, 137.83, 137.74, 133.13, 133.01, 132.95, 132.69, 132.68, 130.38, 130.14, 130.11, 129.99, 129.94, 129.89, 129.78, 129.75, 129.74, 129.45, 128.95, 128.70, 128.67, 128.65, 128.62, 128.55, 128.47, 128.45, 128.41, 128.39, 128.36, 128.26, 128.23, 128.19, 128.15, 128.02, 127.95, 127.93, 127.91, 127.75, 127.66, 127.36, 127.22, 99.53, 97.97, 97.73, 95.81, 93.74, 80.83, 80.40, 80.26, 79.27, 78.40, 78.25, 77.52, 76.58, 76.15, 75.91, 75.74, 75.16, 74.68, 74.20, 74.01, 73.68, 73.58, 73.27, 72.87, 72.19, 71.93, 71.20, 71.16, 70.59, 70.33, 68.69, 68.09, 67.99, 67.33, 67.22, 50.61, 47.18, 46.26, 29.44, 23.56, 18.58, 17.75; HRMS (MALDI-TOF): Calcd for C.sub.148H.sub.149NO.sub.31Na.sup.+ [M+Na].sup.+ 2459.0006. found 2459.0636.
5-Amino-pentanyl α-L-rhamnopyranosyl-(1→3)-β-D-glucopyranosyl-(1→4)-[α-L-rhamnopyranosyl-(1→3)]-α-D-glucopyranosyl-(1→2)-α-D-glucopyranoside (1)
(61) To a solution of fully protected pentasaccharide 34 (23 mg, 9.4 μmol) in THF (1.5 ml) NaOMe (0.5 M, in MeOH, 1 ml) was added and stirred for 12 h. The mixture was neutralized with Amberlite IR 120 (H.sup.+) ion exchange resin, filtered and concentrated. Column chromatography on silica gel (DCM/acetone/MeOH) afforded the de-benzoylated pentasaccharide (16 mg), which was dissolved in a mixture of THF (1 ml) MeOH (1 ml), H.sub.2O (0.7 ml) and AcOH (0.1 ml). The solution was purged with Ar, 10% Pd/C (30 mg) was added and the solution purged with H.sub.2 for 30 min, then stirred under an H.sub.2 atmosphere for 12 h, filtered and concentrated. Size exclusion chromatography on Sephadex LH-20 (MeOH) afforded 1 (5.0 mg, 5.7 μmol, 60%). NMR data is consistent with previously reported..sup.3
EXAMPLE 2
Preparation of PS-1 Substructures
5-Amino-pentanyl D-glucopyranosyl-(1→2)-α-D-glucopyranoside (35)
(62) A solution of protected disaccharide 33 (40 mg, 31 μmol) in a mixture of MeOH (5.0 ml), THF (2.5 ml) H.sub.2O (2.0 ml) and AcOH (0.5 ml) was purged with Ar. After that 10% Pd/C (70 mg) was added and the solution purged with H.sub.2 for 30 min, then stirred under an H.sub.2 atmosphere for 12 h, filtered and concentrated. The crude product was purified by reversed phase solid phase extraction (RP SPE) (Waters Sep-Pak®, C18) to afford 35 (13.3 mg, 31 μmol, 99%). .sup.1H-NMR (600 MHz, D.sub.2O) δ 5.23 (d, J=3.4, 1H, anomeric), 5.16 (d, J=3.6, 1H, anomeric), 4.02-3.80 (m, 8H), 3.75 (app. dd, J=9.9, 3.5, 2H) 3.68-3.61 (m, 2H), 3.53 (app. td, J=9.6, 4.7, 2H), 3.09 (app. t, J=7.5, 2H), 1.81-1.71 (m, 4H, linker), 1.59-1.49 (m, 2H, linker); .sup.13C-NMR (150 MHz, D.sub.2O) δ 98.6 (anomeric), 97.9 (anomeric), 77.7, 75.4, 74.5, 74.4, 74.2, 74.0, 72.3, 72.1, 70.4, 63.3, 63.1, 42.1, 30.6, 29.2, 25.1; HRMS (MALDI-TOF): Calcd for C.sub.17H.sub.33NO.sub.11H.sup.+ [M+H].sup.+ 428.2126. found 428.2147.
5-Amino-pentanyl β-D-glucopyranosyl-(1→4)-α-D-glucopyranosyl-(1→2)-α-D-glucopyranoside (36)
(63) To a solution of protected trisaccharide 33 (60 mg, 31 μmol) in THF (2 ml) NaOMe (0.5 M in MeOH, 0.5 ml) was added and stirred for 4 h. The mixture was neutralized with Amberlite IR 120 (H.sup.+) ion exchange resin, filtered and concentrated. The crude product was dissolved in a mixture of THF (5.0 ml) MeOH (2.5 ml), H.sub.2O (2.0 ml) and AcOH (0.5 ml). The solution was purged with Ar, then 10% Pd/C (30 mg) was added and the solution purged with H.sub.2 for 30 min, then stirred under an H.sub.2 atmosphere for 12 h, filtered and concentrated. Purification by RP SPE (Waters Sep-Pak®, C18) afforded 36 (13.3 mg, 31 μmol, 66%). .sup.1H-NMR (600 MHz, D.sub.2O) δ 5.22 (d, J=3.3, 1H, anomeric α-Glc), 5.15 (d, J=3.6, 1H, anomeric α-Glc), 4.60 (d, J=7.9, 1H, anomeric β-Glc), 4.14-4.08 (m, 1H), 4.03-3.91 (m, 5H), 3.90-3.79 (m, 4H), 3.78-3.72 (m, 3H), 3.71-3.62 (m, 2H), 3.62-3.47 (m, 4H), 3.40 (t, J=8.7, 1H), 3.09 (t, J=7.5, 2H), 1.83-1.72 (m, 4H, linker), 1.59-1.49 (m, 2H, linker). .sup.13C-NMR (150 MHz, D.sub.2O) δ 100.7 (anomeric β-Glc), 94.0 (anomeric α-Glc), 93.4 (anomeric α-Glc), 76.8, 74.2, 73.7, 73.5, 71.3, 69.8, 69.6, 69.5, 69.2, 68.7, 67.7, 67.6, 65.9, 58.8, 57.9, 37.5, 26.1, 24.6, 20.6; HRMS (MALDI-TOF): Calcd for C.sub.23H.sub.43NO.sub.16Na.sup.+ [M+Na].sup.+ 612.2474. found 612.2424.
(2-Methyl-5-tert-butylphenyl) 2-O-benzoyl-4,6-di-O-benzyl-1-thio-β-D-glucopyranoside (40)
(64) A solution of TBAF.3H.sub.2O (1.10 g, 3.48 mmol) and acetic acid (266 μl, 4.64 mmol) in DMF (4 ml) was added to a solution of 29 (430 mg, 0.58 mmol) in DMF (4 ml). The mixture was stirred for 3 days at 35° C. After dilution with Et.sub.2O the phases were separated and the organic phase washed with a 0.1 M HCl solution, saturated aqueous NaHCO.sub.3 solution and brine. The organic phase was then dried over MgSO4, filtered and concentrated. The product 40 was taken directly to the next step.
(2-Methyl-5-tert-butylphenyl) 2,3-di-O-benzoyl-4-O-benzyl-α-L-rhamnopyranosyl-(1→3)2-O-benzoyl-4,6-di-O-benzyl-1-thio-β-D-glucopyranoside (41)
(65) Rhamnosyl-imidate 5 (373 mg, 0.59 mmol) and glucoside 40 (approx. 0.58 mmol) were coevaporated with toluene three times, dried in vacuo and dissolved in anhydrous DCM (3.0 ml). Freshly activated molecular sieves (4 Å) were added and the mixture cooled to −40° C. TMSOTf (10 μl, 53 μmol) was added and the reaction was warmed to −20° C. over 1.5 h. The reaction was quenched with TEA and concentrated. Column chromatography on silica gel (hexanes/ethyl acetate) afforded 41 (490 mg, 0.46 mmol, 79%). [α].sub.D.sup.20=+70.7° (c=1.9, CHCl.sub.3), IR ν.sub.max (film) 2963, 1728, 1602, 1451, 1259, 1090, 1067, 1025 cm.sup.−1; .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 8.02-7.03 (m, 33H), 5.72 (dd, J=9.4, 3.5, 1H), 5.53-5.42 (m, 2H), 5.22 (d, J=1.9, 1H), 4.88 (d, J=10.6, 1H), 4.77-4.47 (m, 6H), 4.24-4.13 (m, 2H), 3.92-3.80 (m, 3H), 3.68-3.59 (m, 2H), 2.18 (s, 3H), 1.25 (s, 9H), 1.08 (d, J=6.2, 3H). .sup.13C-NMR (100 MHz, CDCl.sub.3) δ 149.8, 138.1, 138.0, 133.1, 130.3, 130.0, 129.9, 129.8, 129.7, 129.7, 128.64, 128.57, 128.51, 128.46, 128.42, 128.39, 128.37, 128.32, 128.28, 128.24, 128.20, 128.1, 128.00, 127.97, 127.9, 127.83, 127.80, 127.75, 125.7, 124.4, 97.6, 86.6, 79.3, 77.5, 77.2, 76.8, 75.7, 75.6, 75.0, 74.4, 73.8, 72.0, 71.3, 68.3, 67.9, 31.5, 19.5, 18.0; HRMS (MALDI-TOF): Calcd for C.sub.65H.sub.66O.sub.12SNa.sup.+ [M+Na].sup.+ 1093.4167. found 1093.4159.
N-(Benzyl)benzyloxycarbonyl-5-amino-pentanyl 2,3-di-O-benzoyl-4-O-benzyl-α-L-rhamnopyranosyl-(1→3)2-O-benzoyl-4,6-di-O-benzyl-1-thio-β-D-glucopyranoside (42)
(66) Disaccharide 41 (50 mg, 47 μmol) and 5-aminopentanol (31 mg, 93 μmol) were coevaporated with toluene three times and dried in vacuo. The mixture was dissolved in DCM (3 ml) and NIS (13 mg, 56 μmol) was added and cooled to −20° C. TfOH (0.5 μl, 6 μmol) was added and the mixture was stirred and warmed up to 0° C. in two hours. The reaction was quenched by the addition of aqueous Na.sub.2S.sub.2O.sub.3 and saturated aqueous NaHCO.sub.3. The phases were separated and the aqueous phase was extracted with DCM. The combined organic phases were dried over MgSO.sub.4, filtered and concentrated. The crude product was purified by column chromatography on silica gel (hexanes/ethyl acetates) to afford 42 (52 mg, 43 μmol, 91%). [α].sub.D.sup.20+50.3° (c=2.6, CHCl.sub.3), IR ν.sub.max (film) 3032, 2936, 1730, 1698, 1452, 1265, 1069 cm.sup.−1; .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 8.23-6.80 (m, 40H, aromatic), 5.73 (dd, J=9.4, 3.5, 1H), 5.46 (dd, J=3.4, 1.9, 1H), 5.35 (dd, J=9.2, 7.9, 1H), 5.24 (d, J=1.9, 1H, anomeric Rha), 5.14 (bs, 2H), 4.89 (app. d, J=10.6, 1H), 4.72-4.59 (m, 4H), 4.56-4.35 (m, 4H, anomeric Glc), 4.22-4.12 (m, 2H), 3.91-3.76 (m, 4H), 3.68-3.58 (m, 2H), 3.42-3.33 (m, 1H), 3.05-2.88 (m, 2H), 1.50-1.29 (m, 4H, linker), 1.24-0.98 (m, 5H, linker, Rha CH.sub.3). .sup.13C-NMR (100 MHz, CDCl.sub.3) δ 165.7, 164.8, 138.2, 138.0, 137.6, 133.1, 132.8, 30.0, 129.92, 129.88, 129.8, 129.7, 128.6, 128.5, 128.42, 128.38, 128.36, 128.31, 128.30, 128.2, 128.0, 127.94, 127.93, 127.8, 127.7, 101.1 (anomeric Glc), 97.6 (anomeric Rha), 79.3, 77.8, 76.9, 75.64, 75.59, 74.9, 74.6, 73.8, 71.9, 71.3, 68.9, 68.3, 67.2, 29.2, 23.2, 18.0 (Rha CH.sub.3); HRMS (MALDI-TOF): Calcd for C.sub.74H.sub.75NO.sub.15Na.sup.+ [M+Na].sup.+ 1240.5029. found 1240.4792.
5-Amino-pentanyl α-L-rhamnopyranosyl-(1→3)-β-D-glucopyranoside (38)
(67) To a solution of protected disaccharide 42 (50 mg, 41 μmol) in THF (2 ml) NaOMe (0.5 M in MeOH, 0.5 ml) was added and stirred for 4 h. The mixture was neutralized with Amberlite IR 120 (H.sup.+) ion exchange resin, filtered and concentrated. The crude product was dissolved in a mixture of THF (5.0 ml) MeOH (2.5 ml), H.sub.2O (2.0 ml) and AcOH (0.5 ml). The solution was purged with Ar, then 10% Pd/C (100 mg) was added and the solution purged with H.sub.2 for 30 min, then stirred under an H.sub.2 atmosphere for 12 h, filtered and concentrated. Purification by RP SPE (Waters Sep-Pak®, C18) afforded 38 (15.7 mg, 27 μmol, 78%). .sup.1H-NMR (600 MHz, D.sub.2O) δ 5.20 (s, 1H, anomeric Rha), 4.53 (d, J=8.1, 1H, anomeric Glc), 4.15-4.04 (m, 2H), 4.02-3.96 (m, 2H), 3.85 (app. dd, J=9.7, 3.3, 1H), 3.81-3.73 (m, 2H), 3.66 (app. t, J=8.7, 1H), 3.56-3.49 (m, 3H), 3.44 (t, J=8.7, 1H), 3.08 (app. t, J=7.5, 2H), 1.75 (tt, J=14.6, 7.2, 4H, linker), 1.57-1.49 (m, 2H, linker), 1.32 (d, J=6.3, 3H, Rha CH.sub.3); .sup.13C-NMR (150 MHz, D.sub.2O) δ 100.0 (anomeric Glc), 99.1 (anomeric Rha), 80.3, 73.9, 71.8, 70.0, 68.4, 68.2, 68.1, 66.9, 66.2, 58.8, 37.4, 26.2, 24.4, 20.1, 14.5 (Rha CH.sub.3); HRMS (MALDI-TOF): Calcd for C.sub.17H.sub.33NO.sub.10Na.sup.+ [M+Na].sup.+ 434.1997. found 434.1975.
N-(Benzyl)benzyloxycarbonyl-5-amino-pentanyl 2,6-di-O-benzyl-3-O-(4-bromo)benzyl-4-O-levulinoyl-1-thio-β-D-glucopyranoside (43)
(68) Thioglucoside 27 (300 mg, 0.38 mmol) and 5-aminopentanol (200 mg, 0.61 mmol) were coevaporated with toluene three times and dried in vacuo. The mixture was dissolved in Ether (4 ml) and Dioxane (4 ml), NIS (103 mg, 0.46 mmol) was added and cooled to −10° C. TfOH (4 μl, 46 μmol) was added and the mixture was stirred and warmed up to 0° C. in three hours. The reaction was quenched by the addition of aqueous Na.sub.2S.sub.2O.sub.3 and saturated aqueous NaHCO.sub.3, The phases were separated and the aqueous phase was extracted with DCM. The combined organic phases were dried over MgSO.sub.4, filtered and concentrated. The crude product was purified by column chromatography on silica gel (hexanes/ethyl acetates) to afford 43 (140 mg, 0.15 mmol, 39%). [α].sub.D.sup.20=+22.0° (c=3.4, CHCl.sub.3), IR ν.sub.max (film) 2920, 1743, 1697, 1454, 1420, 1360, 1208, 1153, 1069, 1038 cm.sup.−1; .sup.1H-NMR (400 MHz, CDCl.sub.3) δ7.696.92 (m, 24H, ar), 5.22-5.15 (m, 2H), 5.09-5.03 (m, 1H), 4.81 (app. d, J=11.9, 1H), 4.76-4.68 (m, 2H, anomeric), 4.63-4.56 (m, 2H), 4.54-4.46 (m, 4H), 3.89 (app. t, J=9.4, 1H), 3.84-3.78 (m, 1H), 3.62-3.45 (m, 4H), 3.38-3.18 (m, 3H), 2.66-2.53 (m, 2H), 2.43-2.29 (m, 2H), 2.13 (s, 3H, Lev CH.sub.3), 1.66-1.48 (m, 4H, linker), 1.38-1.27 (m, 2H, linker); .sup.13C-NMR (100 MHz, CDCl.sub.3) δ 206.3 (Lev carbonyl), 171.6, 138.2, 138.1, 138.0, 131.4, 129.6, 129.4, 128.7, 128.5, 128.3, 128.1, 128.03, 127.99, 127.9, 127.6, 127.4, 121.3, 96.9 (anomeric), 79.8, 79.6, 74.3, 73.7, 73.2, 70.9, 69.0, 68.9, 68.3, 67.3, 37.8, 29.9 (Lev CH.sub.3), 29.2, 28.0, 23.6; HRMS (MALDI-TOF): Calcd for C.sub.52H.sub.58BrNO.sub.10Na.sup.+ [M+Na].sup.+ 958.3134. found 958.3112.
N-(Benzyl)benzyloxycarbonyl-5-amino-pentanyl 2,6-di-O-benzyl-3-O-(4-bromo)benzyl-1-thio-β-D-glucopyranoside (44)
(69) To a solution of 43 (140 mg, 0.15 mmol) in DCM (5.0 ml) hydrazine hydrate (26 μl, 0.54 mmol) dissolved in AcOH (0.4 ml) and pyridine (0.6 ml) was added and the solution stirred for 1 h. The reaction was then quenched by the addition of acetone and concentrated. Column chromatography on silica gel (hexanes/ethyl acetate) afforded 44 (102 mg, 0.12 mmol, 810). [α].sub.D.sup.20=+24.3° (c=4.2, CHCl.sub.3), IR ν.sub.max (film) 3454, 3031, 2920, 1696, 1454, 1422, 1229, 1055 cm.sup.−1; .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 7.48-7.04 (m, 24H, Ar), 5.16-5.09 (m, 2H), 4.86 (app. d, J=11.7, 1H), 4.70-4.43 (m, 8H), 3.75-3.55 (m, 6H), 3.45 (app. dd, J=9.5, 3.6, 1H), 3.32-3.14 (m, 3H), 1.59-1.44 (m, 4H, linker), 1.33-1.23 (m, 2H, linker); .sup.13C-NMR (100 MHz, CDCl.sub.3) δ 138.3, 138.1, 138.0, 131.6, 129.5, 128.6, 128.52, 128.47, 128.02, 127.98, 127.9, 127.8, 127.7, 127.4, 121.6, 96.9 (anomeric), 81.7, 79.8, 74.6, 73.7, 72.9, 71.4, 70.1, 69.8, 68.1, 67.3, 50.4, 47.3, 29.2, 27.7, 23.7; HRMS (MALDI-TOF): Calcd for C.sub.47H.sub.52BrNO.sub.8Na.sup.+ [M+Na].sup.+ 860.2769. found 860.2508.
N-(Benzyl)benzyloxycarbonyl-5-amino-pentanyl 2,3-di-O-benzoyl-4-O-benzyl-α-L-rhamnopyranosyl-(1→3)-2-O-benzoyl-4,6-O-benzyl-β-D-glucopyranosyl-(1→4)-2,6-di-O-benzyl-3-O-(4-bromo)benzyl-α-D-glucopyranoside (45)
(70) Disaccharide 41 (144 mg, 0.13 mmol) and glucoside 44 (102 mg, 0.12 mmol) were coevaporated with toluene three times and dried in vacuo. The mixture was dissolved in DCM (4 ml) and NIS (36 mg, 0.16 mmol) was added and cooled to −20° C. TfOH (1.4 μl, 16 μmol) was added and the mixture was stirred and warmed up to 0° C. in two hours. The reaction was quenched by the addition of aqueous Na.sub.2S.sub.2O.sub.3 and saturated aqueous NaHCO.sub.3. The phases were separated and the aqueous phase was extracted with DCM. The combined organic phases were dried over MgSO.sub.4, filtered and concentrated. The crude product was purified by column chromatography on silica gel (hexanes/ethyl acetates) to afford 45 (200 mg, 0.12 mmol, 95%). [α].sub.D.sup.20=+36.9° (c=5.2, CHCl.sub.3), IR ν.sub.max (film) 3031, 2866, 1730, 1698, 1602, 1452, 1262, 1092 cm.sup.−1; .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 8.31-6.72 (m, 54H, Ar), 5.73 (app. dd, J=9.4, 3.4, 1H), 5.44 (app. dd, J=3.4, 1.9, 1H), 5.37 (app. dd, J=9.3, 8.1, 1H), 5.21 (bs, 2H), 5.17-5.09 (m, 2H), 4.88 (app. d, J=10.9, 1H), 4.76-4.39 (m, 13H), 4.31 (app. d, J=12.2, 1H), 4.19 (app. dd, J=9.5, 6.1, 1H), 4.03-3.63 (m, 9H), 3.49-3.42 (m, 3H), 3.37-3.15 (m, 4H), 1.59-1.40 (m, 4H), 1.28-1.11 (m, 5H); .sup.13C-NMR (100 MHz, CDCl.sub.3) δ 165.2, 164.6, 164.5, 138.9, 138.5, 138.2, 138.0, 137.89, 137.87, 137.6, 133.1, 133.0, 132.9, 131.1, 129.9, 129.8, 129.7, 129.63, 129.59, 129.4, 129.2, 128.7, 128.6, 128.5, 128.43, 128.35, 128.3, 128.24, 128.19, 128.14, 128.08, 128.0, 127.90, 127.89, 127.74, 127.67, 127.61, 127.55, 127.3, 120.7, 100.3 (anomeric), 97.7 (anomeric), 96.9 (anomeric), 80.3, 79.1, 78.0, 77.4, 76.7, 75.6, 75.2, 74.9, 74.8, 74.5, 73.6, 73.5, 73.1, 71.9, 71.1, 69.7, 68.8, 68.3, 68.0, 67.7, 67.2, 29.0, 23.3, 17.9 (Rha CH.sub.3); HRMS (MALDI-TOF): Calcd for C.sub.101H.sub.102BrNO.sub.20Na.sup.+ [M+Na].sup.+ 1750.6071. found 1759.5921.
5-Amino-pentanyl α-L-rhamnopyranosyl-(1→3)-β-D-glucopyranosyl-(1→4)-α-D-glucopyranoside (37)
(71) To a solution of protected trisaccharide 45 (61 mg, 35 μmol) in THF (2 ml) NaOMe (0.5 M in MeOH, 0.5 ml) was added and stirred for 4 h. The mixture was neutralized with Amberlite IR 120 (H.sup.+) ion exchange resin, filtered and concentrated. The crude product was dissolved in a mixture of THF (5.0 ml) MeOH (2.5 ml), H.sub.2O (2.0 ml) and AcOH (0.5 ml). The solution was purged with Ar, then 10% Pd/C (100 mg) was added and the solution purged with H.sub.2 for 30 min, then stirred under an H.sub.2 atmosphere for 12 h, filtered and concentrated. Purification by RP SPE (Waters Sep-Pak®, C18) afforded 37 (12.5 mg, 30 μmol, 75%). .sup.1H-NMR (600 MHz, D.sub.2O) δ 5.21 (s, 1H, anomeric Rha), 4.99 (d, J=2.9, 1H, anomeric α-Glc), 4.61 (d, J=8.0, 1H, anomeric β-Glc), 4.15-4.05 (m, 2H), 4.02-3.97 (m, 2H), 3.93-3.79 (m, 6H), 3.73-3.66 (m, 3H), 3.64-3.49 (m, 5H), 3.09 (t, J=7.1, 2H), 1.81-1.71 (m, 4H, linker), 1.59-1.50 (m, 2H, linker), 1.33 (d, J=6.0, 3H, Rha CH.sub.3); .sup.13C-NMR (150 MHz, D.sub.2O) δ 102.9 (anomeric Rha), 101.7 (anomeric β-Glc), 98.4 (anomeric α-Glc), 82.7, 79.7, 76.5, 74.5, 72.6, 72.4, 71.6, 71.1, 71.0, 70.8, 69.4, 68.6, 68.5, 61.2, 60.6, 40.0, 28.6, 27.1, 23.0, 17.1. (Rha CH.sub.3); HRMS (MALDI-TOF): Calcd for C.sub.23H.sub.43BrNO.sub.25Na.sup.+ [M+Na].sup.+ 596.2525. found 596.2540.
N-(Benzyl)benzyloxycarbonyl-5-amino-pentanyl 2,3-di-O-benzoyl-4-O-benzyl-α-L-rhamnopyranoside (46)
(72) Rhamnoside-imidate (127 mg, 0.20 mmol) and 5-aminopentanol (160 mg, 0.49 mmol) were coevaporated with toluene three times, dried in vacuo and dissolved in anhydrous DCM (3 ml). Freshly activated molecular sieves (4 Å) were added and the mixture cooled to −30° C. TMSOTf (3.6 μl, 20 μmol) was added and the reaction was warmed to −20° C. over 1 h. The reaction was quenched with TEA and concentrated. Column chromatography on silica gel (hexanes/ethyl acetate) afforded 46 (145 mg, 0.19 mmol, 94%). [α].sub.D.sup.20+54.1° (c=2.6, CHCl.sub.3), IR ν.sub.max (film) 2963, 1727, 1260, 1018 cm.sup.−1; .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 8.28-7.00 (m, 25H, Ar), 5.73 (app. dd, J=9.6, 3.4, 1H), 5.59 (bs, 1H), 5.19 (app d, J=11.3, 2H), 4.87 (bs, 1H, anomeric), 4.68 (app. dd, J=28.1, 10.9, 2H), 4.53 (bs, 2H), 3.96 (bs, 1H), 3.79 (app. t, J=9.5, 1H), 3.75-3.61 (m, 1H), 3.48-3.21 (m, 3H), 1.65-1.51 (m, 4H), 1.45-1.27 (m, 5H); .sup.13C-NMR (100 MHz, CDCl.sub.3) δ 165.6, 165.5, 138.1, 137.8, 133.4, 133.2, 130.0, 129.9, 129.7, 128.7, 128.6, 128.47, 128.46, 128.2, 127.99, 127.95, 127.4, 97.5 (anomeric), 79.3, 75.3, 72.6, 71.5, 68.0, 67.8, 67.3, 29.3, 23.6, 18.3; HRMS (MALDI-TOF): Calcd for C.sub.47H.sub.49NO.sub.9Na.sup.+ [M+Na].sup.+ 794.3300. found 794.3264.
5-Amino-pentanyl α-L-rhamnopyranoside (39)
(73) To a solution of protected rhamnoside 46 (145 mg, 0.19 mmol) in THF (4 ml) NaOMe (0.5 M in MeOH, 0.5 ml) was added and stirred for 4 h. The mixture was neutralized with Amberlite IR 120 (H.sup.+) ion exchange resin, filtered and concentrated. The crude product was dissolved in a mixture of THF (10 ml) MeOH (5 ml), H.sub.2O (4 ml) and AcOH (1 ml). The solution was purged with Ar, then 10% Pd/C (300 mg) was added and the solution purged with H.sub.2 for 30 min, then stirred under an H.sub.2 atmosphere for 12 h, filtered and concentrated. Purification by RP SPE (Waters Sep-Pak®, C18) afforded 39 (44 mg, 0.18 mmol, 94%). .sup.1H-NMR (600 MHz, D.sub.2O) δ 4.85 (s, 1H, anomeric Rha), 4.01-3.96 (m, 1H), 3.81-3.70 (m, 3H), 3.62-3.57 (m, 1H), 3.50 (app. t, J=9.6, 1H), 3.11-3.03 (m, 2H), 1.78-1.67 (m, 4H, linker), 1.56-1.46 (m, 2H), 1.34 (d, J=6.3, 3H, Rha CH.sub.3). .sup.13C-NMR (150 MHz, D.sub.2O) δ 98.3 (anomeric), 70.6, 70.0, 68.8, 67.1, 66.1, 38.0, 26.6, 25.1, 21.0, 15.2 (Rha CH.sub.3); HRMS (MALDI-TOF): Calcd for C.sub.11H.sub.23NO.sub.5Na.sup.+ [M+Na].sup.+ 272.1468. found 272.1433.
EXAMPLE 3
Preparation and Characterization of an Pentasaccharide-Protein Conjugate
(74) Polysaccharide vaccines provoke exclusively a T-cell independent immune response and do not induce an immunoglobulin class switch. The synthetic repeating unit 1 of the Clostridium difficile glycopolymer PS-I was conjugated to the protein carrier Crm.sub.197. The detoxified diphtheria toxoid Crm.sub.197 was chosen as a carrier since it is an approved constituent of licensed vaccines (Barocchi et al. (2007), Vaccine 25, 2963-73).
(75) Conjugations
(76) A) To a solution of di(N-succinimidyl) adipate (5.8 mg, 17 μmol) in DMSO (250 μl) and NEt.sub.3 (20 μl) pentasaccharide 1 (500 μg, 0.57 μmol) dissolved in DMSO (250 μl) was added dropwise. The solution was stirred for 2 h, diluted with phosphate buffer (1.0 ml, 100 μm, pH 7.5) and extracted with CHCl.sub.3. CRM.sub.197 (rDNA) (250 μl, 250 μg, Pfenex Inc (USA)) was added to the aqueous layer and stirred for 5 h. Conjugate 1a was desalted and concentrated. An average load of 3.6 pentasaccharide units per protein was determined by MALDI-TOF MS, SEC-HPLC and SDS PAGE confirmed modification of the protein (
(77) B) First, the primary amine group of the linker moiety of PS-I pentasaccharide 1 was reacted with one of the ester groups of the spacer molecule di(N-succinimidyl) adipate in water-free DMSO (12.7 mg in 120 μl) in the presence of 10 μl triethylamine at room temperature over 2 hours, with the spacer used in 10-fold molar excess to avoid dimer formation. After addition of 400 μL 0.1 M Na-phosphate buffer, pH 7.4, unreacted spacer molecules were removed by chloroform extraction. The remaining ester group of the spacer moiety was then reacted with the ϵ-amino groups of lysine residues on the CRM.sub.197 protein (Pfenex) in 0.1 M Na-phosphate buffer, pH 7.4, at room temperature over 12 hours (
(78) Successful conjugation was confirmed by SDS-PAGE as shown in
(79) The oligosaccharide/CRM.sub.197 ratio was determined by MALDI-TOF MS. The mass analysis of CRM.sub.197 yielded a m/z ion at 58.2 kDa. The mass analysis of the conjugate yielded a major m/z ion at 67.7 kDa and further peaks ˜1000 Da apart, corresponding to conjugates of different valencies (
(80) Knowing the protein concentration of the conjugate, as determined by bicinchoninic acid (BCA) assay, and the average sugar loading, the carbohydrate content was calculated to 300±46 μg/mL (mean±SD) and verified by colorimetric anthrone assay (302±76 μg/mL), an approved method for the carbohydrate determination of the licensed pneumococcal conjugate vaccine Prevenar (Pfizer).
(81) SDS-PAGE
(82) Pentasaccharide 1-CRM.sub.197 conjugate and unconjugated CRM.sub.197 were dissolved in Lämmli buffer (0.125 M Tris, 20% (v/v) glycerol, 4% (w/v) SDS, 5% (v/v) beta-mercaptoethanol, bromophenol, pH 6.8) and boiled at 95° C. for 5 minutes. Samples were run in 10% polyacrylamide gels and stained with 0.025% (w/v) Coomassie Brilliant blue R-250 in an aqueous solution containing 40% (v/v) methanol and 7% (v/v) acetic acid.
(83) MALDI-TOF Mass Spectrometry
(84) Conjugation was confirmed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) using an Autoflex™ Speed instrument (Bruker Daltonics, Bremen, Germany). The mass spectrometer was operated in positive linear mode. Spectra were acquired over an m/z range from 50,000 to 85,000 Da and data was analyzed with the FlexAnalysis software provided with the instrument. 2′,4′-dihydroxyacetonephenone (DHAP) was used as matrix, samples were spotted using the dried droplet technique.
(85) Anthrone Assay
(86) Anthrone assays were performed in 96-well format in a modified assay according to Leyva et al., Biologicals 36:134-141, 2008. Briefly, 75 μL of anthrone reagent (0.1% (w/v) in concentrated sulfuric acid) was added to each well of a 96-well microtiter plate containing 25 μL of standard solutions, sample dilutions and blank. Plates were first placed at 4° C. for 10 minutes, then incubated at 100° C. for 20 minutes, and cooled down at room temperature for 20 minutes. Absorbance at 579 nm was determined in a microplate reader. Colorimetric response was compared to a standard curve based on glucose and rhamnose in a 3:2 molar ratio.
EXAMPLE 4
Immunization and Monoclonal Antibodies
(87) To test the immunogenicity of the PS-I pentasaccharide hapten, three groups of six female C57BL/6 mice each were immunized subcutaneously (s.c.) with conjugate (one group without adjuvant, one group with Freund's adjuvant, one group with Alum adjuvant). Each mouse received an amount of conjugate corresponding to 3 μg PS-I pentasaccharide 1 antigen. Initial immunizations (priming) was followed by an immunization after two weeks (boosting). Sera were collected in one-week intervals. IgG antibody responses were evaluated by microarray. PS-I pentasaccharide 1 in three different concentrations (1, 0.5 and 0.1 mM), CRM.sub.197 (1, 0.5 and 0.1 μM) and bovine serum albumin (BSA)-spacer-GlcNAc conjugate (1, 0.5 and 0.1 μM) were spotted in triplicate onto the surface of the microarray slides (N-hydroxysuccinimide ester-activated glass slides (CodeLink)) as shown in
(88) PS-I pentasaccharide-specific IgG antibody responses were identified in pooled sera of three groups (each n=6) of immunized mice after priming, and more pronounced after boosting (week 3), as determined by microarray analysis (
(89) IgG antibody responses were quantified by determination of the fluorescence intensity values using the sera of individual mice. While the conjugate already showed immunogenicity without adjuvant (
(90) As an IgG-specific detection antibody, Anti-Mouse IgG (whole molecule)-FITC (Sigma) was used in the tests of
(91) To get an insight into the subclasses of IgG antibodies raised against PS-I pentasaccharide, microarray analysis with pooled sera using subclass-specific detection antibodies against IgG1, IgG2a and IgG3 was performed.
(92)
(93) As evident from
(94) To assess whether antibodies raised with PS-I pentasaccharide antigen 1 recognize substructures of the antigen as well, which allows to define the minimal epitope, microarray slides with substructures 35-39 in addition to 1 were prepared (
(95)
(96)
(97)
(98) As shown in
(99) Monoclonal antibodies were generated with the traditional hybridoma technique [Köhler and Milstein, 1975]. Three monoclonal antibodies (mAbs), 2C5, 10A1 and 10D6, were selected for evaluation with deletion sequence microarray and isotype-specific detection antibodies. All three mABs showed identical patterns on the microarray, exclusively bound to PS-I pentasaccharide 1 but none of the substructures, and were of the IgG1 subtype (
(100)
(101) Immunizations
(102) Six to eight-weeks old female C57BL/6 mice were immunized s.c. with conjugate corresponding to 3 μg PS-I pentasaccharide 1 with Freund's (priming immunizations with Freund's Complete Adjuvant, boosting immunizations with Freund's Incomplete Adjuvant, both Sigma) or Aluminium Hydroxide Gel Adjuvant (Brenntag Biosector, Frederikssund, Denmark), or without adjuvant. Mice received boosting injections after 2 weeks. For all immunizations, antigen was diluted in sterile PBS to a total injection volume of 100 μL per mouse. Blood was collected in one-week intervals via the tail vein and erythrocytes separated from serum by centrifugation. Serum antibody responses were analyzed by microarray. One mouse of the Alum group received a second boosting injection s.c. at week 5 after first immunization, and, prior to being sacrificed, three final boosting injections via the intraperitoneal (i.p.) route, on three consecutive days at week 7.
(103) Preparation of Microarrays
(104) Oligosaccharides bearing an amine linker, or proteins, were dissolved in sodium phosphate buffer (50 mM, pH 8.5) and printed robotically using a piezoelectric spotting device (S11, Scienion, Berlin, Germany) onto NHS-activated glass slides (CodeLink). Slides were incubated in a humid chamber to complete reaction for 24 hours and stored in an anhydrous environment. Prior to the experiment, remaining succinimidyl groups were quenched by incubating slides in 100 mM ethanolamine in sodium phosphate buffer (pH 9, 50 mM) for 1 hour at 50° C. Slides were rinsed three times with deionized water and dried by centrifugation.
(105) Microarray Binding Assays
(106) The quenched array slides were blocked for 1 hour with 1% (w/v) BSA in PBS, then washed three times with PBS and dried by centrifugation. A FlexWell 64 (Grace Bio-Labs, Bend, Oreg., USA) grid was applied to the slides. Resulting 64 wells were used for 64 individual experiments. Slides were incubated with sera dilutions or hybridoma supernatants (all dilutions were prepared with PBS) for 1 hour at room temperature in a humid chamber, washed three times with PBS-Tween-20 (0.1% v/v) and dried by centrifugation. Then, slides were incubated with fluorescence-labeled detection antibody diluted in 1% BSA in PBS (w/v) for 1 hour at room temperature in a humid chamber. Slides were washed three times with PBS-Tween-20 (0.1% v/v) and rinsed once with deionized water and dried by centrifugation. Slides were scanned with a GenePix 4300A scanner (Molecular Devices) using the GenePix Pro 7 software. Detection antibodies used were Anti-Mouse IgG (whole molecule)-FITC (Sigma), Alexa Fluor 635 Goat Anti-Mouse IgG (H+L) (Life Technologies) and Alexa Fluor 594 Goat Anti-Mouse IgG1 (γ1) (Life Technologies) in 1:400 dilutions, as well as Alexa Fluor 647 Goat Anti-Mouse IgG2a (γ2a) and Alexa Fluor 488 Goat Anti-Mouse IgG3 (γ3) (Life Technologies) in 1:200 dilutions.
(107) Monoclonal Antibodies
(108) Monoclonal antibodies (mABs) were generated using the standard method by Köhler and Milstein, 1975. Briefly, spleenocytes of one mouse were fused with 10.sup.8 mouse myeloma cells in the presence of 50% PEG 1500. Fused cells were selected with complete growth medium (IMDM supplemented with 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, 24 μM beta-mercaptoethanol, 100 μM hypoxanthine, 16 μM thymidine, non-essential amino acids, 100 U/mL penicillin, 100 μg/mL streptomycin, 50 μg/mL gentamycin, 10% hybridoma cloning supplement (BM Condimed H1, Roche)) with 0.4 μM aminopterin. Cells were maintained at 37° C. at 5% CO.sub.2. Hybridoma cells were subjected to three consecutive subcloning steps by limited dilution. Clones producing antibodies against PS-I pentasaccharide were identified by microarray analysis.
EXAMPLE 5
Evaluation of the Protective Effects of the Monoclonal Antibodies Directed Against C. difficile PS-I in a Murine Model
(109) Purified monoclonal antibodies 2C5 and 10D6 were tested for their ability to prevent experimental C. difficile disease in mice. Mice provide an established experimental disease model for C. difficile infection (Buffie C G, et al. 2012. Profound alterations of intestinal microbiota following a single dose of clindamycin results in sustained susceptibility to Clostridium difficile-induced colitis. Infect. Immun. 80: 62-73). The animals are made susceptible to infection by treatment with the antibiotic clindamycin followed by oral challenge with C. difficile bacteria obtained from bacterial culture. As bacterial strain, the C. difficile strain M68 PCR ribotype 017 was used (Drudy D, et al. 2007. Isolation and characterisation of toxin A-negative, toxin B-positive Clostridium difficile in Dublin, Ireland. Clin. Microbiol. Infect. 13: 298-304). Mice challenged with an inoculum corresponding to 108 colony-forming units (CFU) develop symptoms of the disease such as inflammation of the colon (colitis). Intestinal colonization with C. difficile bacteria that is detectable in stool samples serves as read-out for the degree of infection (
(110) To assess the efficacy of 2C5 and 10D6 in preventing C. difficile colitis, groups of female C57BL/6 mice received three consecutive doses of the antibodies at outlined in