(2R,3S,4S,5S,6S)-2-(hydroxymethyl)-6-methoxyoxane-3,4,5-triol

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: A ca. 2:1 mixture of exo- and endo-isomers of bis-[(2,3-O-), (4,6-O-)]benzylidene-α-D-mannopyranoside (Carbohydrates, Ed. Collins, P. M., Chapman and Hall, New York, 1987, p. 350) was prepared by reaction of methyl α-D-mannopyranoside with 2.2 eq of α,α-dimethoxytoluene and catalytic p-toluenesulfonic acid in acetonitrile. This compound was treated with NaBH4 and HCl (Garegg, P. J.; Hultberg, H. Carbohydrate Res. 1981, 93, C10) to provide a mixture of products from which the methyl 2,6-O-benzyl-α-D-mannopyranoside was isolated by flash chromatography. The assignment of this isomer was confirmed by 1H decoupling experiments on the corresponding bis-(3,4-O-diphenylphosphino) derivative (ligand VA). 1H NMR δ7.42-7.24 (m, 10), 4.81 (d, 1, J=1 Hz), 4.75-4.54 (m, 4) 3.78-3.71 (m, 6), 3.36 (s, 3), 2.83 (bs, 1), 2.43 (bs, 1). A ca. 2:1 mixture of exo- and 1981, 93, C10) to provide was isolated by flash chromatography

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: A reaction mixture (100 mL, 100 mM Tris, pH 7.5, 10 mM MgCl2, 10 mM MnCl2, 5 mM EDTA, 5 ThM NaN3, 1 mm ATPe 0.01 mM theophylline and 0.03 mM 2, 3-dimercaptopropanol) containing α-methyl-D-mannopyranoside (0.4 g, 20 M), Man-1-P (about 1.5 g, about 50 mM), PEP (1.0 g, 42.7 HM), GDP (30 mg, 1 mM), dried yeast cells (0.4 g), pyruvate kinase PK (300 U), recombinant α1,2-ManT (1.2 U) and inorganic pyrophosphatase (2 U) was slightly stirred at room temperature for 72 hours, then centrifuged. The supernatant was lyophilized and extracted with methanol. After removal of the methanol, the product was purified by silica gel column chromatography (CHCl :CH3OH:H2O=6: 3:0.5, v/v/v) to afford Manα1, 2ManαOMe (Compound 3) (96 mg) in 38 percent overall yield based on consumed α-methyl-D-mannopyranoside. 1H NMR (D2O) δ3.41 (s, 3H), 3.60-3.80 (m, 6H), 3.86 (dd, 1H, J=3.5, 10 Hz), 3.88 (dd, 1H, J=3.5, 10 Hz), 3.90 (d, 1H, d, J=1.5 Hz), 3.92 (d, 1H, J=l Hz), 3.97 (dd, 1H, J=2, 3.5 Hz), 4.08 (dd, 2H, J=2, 3.5 Hz), 5.02 (d, 1H, d, J=2 Hz), 5 .04 (d, 1H, J=2.0 Hz). 13C {1H} NMR (D2O) δ54.90, 61.01, 61.24, 66.98, 67.03, 70.02, 70.29, 70.37, 72.63, 73.40, 78.62, 99.40, 102.43. HRMS calculated for C13H24O11Na+ (M+Na+) 379.1216, found 379.1220. dried yeast cells (0.4 g), pyruvate kinase PK (300 U), recombinant α1,2-ManT (1.2 U) and inorganic pyrophosphatase (2 U) extracted with methanol After removal of the methanol the product was purified by silica gel column chromatography (CHCl

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: To a stirred solution of (2R,3S,4S,5S,6S)-2-(hydroxymethyl)-6-methoxytetrahydro-2H-pyran-3,4,5-triol (15.0 g, 77.2 mmol, 1.0 equiv., commercially available Sigma Aldrich) in dry DMF (250 mL) was added sodium hydride (14.8 g, 309 mmol, 4.0 equiv) at 0° C. under nitrogen atmosphere. The reaction mixture was stirred at the same temperature for 30 minutes and then benzylbromide (38 mL, 309 mmol, 4.0 equiv) was added dropwise. The reaction mixture was stirred at room temperature for 16 hours. The reaction was quenched with MeOH (50 mL) at 0° C. and concentrated in vacuo. Ice cold water (80 mL) was added, the mixture was extracted with EtOAc (2×250 mL), and the combined organic layers were washed with saturated NaHCO3 solution (3×60 mL), dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The material thus obtained was purified by silica gel chromatography, eluting with 20% EtOAc in petroleum ether to afford (2R,3R,4S,5S,6S)-3,4,5-tris(benzyloxy)-2-((benzyloxy)methyl)-6-methoxytetrahydro-2H-pyran as a pale yellow oil (28 g, 65%). 1H NMR (400 MHz, CDCl3) δ 7.47-7.24 (m, 18H), 7.25-7.15 (m, 2H), 4.92 (d, J=10.8 Hz, 1H), 4.79 (dd, J=17.2, 2.2 Hz, 3H), 4.70 (d, J=12.1 Hz, 1H), 4.67-4.50 (m, 4H), 4.01 (t, J=9.1 Hz, 1H), 3.92 (dd, J=9.3, 3.1 Hz, 1H), 3.86-3.72 (m, 4H), 3.36 (s, 3H). ESI (M+H2O) 572.0, calc for C35H38O6 554.3. The reaction mixture was stirred at room temperature for 16 hours The reaction was quenched with MeOH (50 mL) at 0° C. concentrated in vacuo Ice cold water (80 mL) was added the mixture was extracted with EtOAc (2×250 mL) the combined organic layers were washed with saturated NaHCO3 solution (3×60 mL) dried over anhydrous sodium sulfate filtered concentrated in vacuo The material thus obtained was purified by silica gel chromatography eluting with 20% EtOAc in petroleum ether

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: To a stirred solution of (2R,3S,4S,5S,6S)-2-(hydroxymethyl)-6-methoxytetrahydro-2H-pyran-3,4,5-triol (15.0 g, 77.2 mmol, 1.0 equiv., commercially available Sigma Aldrich) in dry DMF (250 mL) was added sodium hydride (14.8 g, 309 mmol, 4.0 equiv) at 0° C. under nitrogen atmosphere. The reaction mixture was stirred at the same temperature for 30 minutes and then benzylbromide (38 mL, 309 mmol, 4.0 equiv) was added dropwise. The reaction mixture was stirred at room temperature for 16 hours. The reaction was quenched with MeOH (50 mL) at 0° C. and concentrated in vacuo. Ice cold water (80 mL) was added, the mixture was extracted with EtOAc (2×250 mL), and the combined organic layers were washed with saturated NaHCO3 solution (3×60 mL), dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The material thus obtained was purified by silica gel chromatography, eluting with 20% EtOAc in petroleum ether to afford (2R,3R,4S,5S,6S)-3,4,5-tris(benzyloxy)-2-((benzyloxy)methyl)-6-methoxytetrahydro-2H-pyran as a pale yellow oil (28 g, 65%). 1H NMR (400 MHz, CDCl3) δ 7.47-7.24 (m, 18H), 7.25-7.15 (m, 2H), 4.92 (d, J=10.8 Hz, 1H), 4.79 (dd, J=17.2, 2.2 Hz, 3H), 4.70 (d, J=12.1 Hz, 1H), 4.67-4.50 (m, 4H), 4.01 (t, J=9.1 Hz, 1H), 3.92 (dd, J=9.3, 3.1 Hz, 1H), 3.86-3.72 (m, 4H), 3.36 (s, 3H). ESI (M+H2O) 572.0, calc for C35H38O6 554.3. The reaction mixture was stirred at room temperature for 16 hours The reaction was quenched with MeOH (50 mL) at 0° C. concentrated in vacuo Ice cold water (80 mL) was added the mixture was extracted with EtOAc (2×250 mL) the combined organic layers were washed with saturated NaHCO3 solution (3×60 mL) dried over anhydrous sodium sulfate filtered concentrated in vacuo The material thus obtained was purified by silica gel chromatography eluting with 20% EtOAc in petroleum ether

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: A ca. 2:1 mixture of exo- and endo-isomers of bis-[(2,3-O-), (4,6-O-)]benzylidene-α-D-mannopyranoside (Carbohydrates, Ed. Collins, P. M., Chapman and Hall, New York, 1987, p. 350) was prepared by reaction of methyl α-D-mannopyranoside with 2.2 eq of α,α-dimethoxytoluene and catalytic p-toluenesulfonic acid in acetonitrile. This compound was treated with NaBH4 and HCl (Garegg, P. J.; Hultberg, H. Carbohydrate Res. 1981, 93, C10) to provide a mixture of products from which the methyl 2,6-O-benzyl-α-D-mannopyranoside was isolated by flash chromatography. The assignment of this isomer was confirmed by 1H decoupling experiments on the corresponding bis-(3,4-O-diphenylphosphino) derivative (ligand VA). 1H NMR δ7.42-7.24 (m, 10), 4.81 (d, 1, J=1 Hz), 4.75-4.54 (m, 4) 3.78-3.71 (m, 6), 3.36 (s, 3), 2.83 (bs, 1), 2.43 (bs, 1). A ca. 2:1 mixture of exo- and 1981, 93, C10) to provide was isolated by flash chromatography

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: A mixture of methyl α-D-mannopyranoside (Compound I, 50 g, 258 mmoles), α,α-dimethoxytoluene (92 g, 600 mmoles), and anhydrous p-toluene-sulfonic acid (1 g) in N,N-dimethylformamide (300 ml), in a 1-liter flask fitted with an air condenser attached to a water aspirator, was stirred magnetically and heated in an oil bath for 3 hours at 65°-75° C. None of the starting Compound I remained after this time (t.l.c., 4:1 ether-petroleum ether). The mixture was poured with vigorous stirring into 1 liter of ice-water containing sodium hydrogen carbonate (30 g). The resultant precipitate was filtered off, resuspended in ice-water, filtered off again, and dried in air and finally in vacuo over phosphorus pentaoxide; yield 90.6 g (95%), m.p. 120°-160° C, [α]D23 -33° (c 1, chloroform). in a 1-liter flask fitted with an air condenser heated in an oil bath for 3 hours at 65°-75° C The mixture was poured The resultant precipitate was filtered off filtered off again dried in air and finally in vacuo over phosphorus pentaoxide

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: A mixture of methyl α-D-mannopyranoside (Compound I, 50 g, 258 mmoles), α,α-dimethoxytoluene (92 g, 600 mmoles), and anhydrous p-toluene-sulfonic acid (1 g) in N,N-dimethylformamide (300 ml), in a 1-liter flask fitted with an air condenser attached to a water aspirator, was stirred magnetically and heated in an oil bath for 3 hours at 65°-75° C. None of the starting Compound I remained after this time (t.l.c., 4:1 ether-petroleum ether). The mixture was poured with vigorous stirring into 1 liter of ice-water containing sodium hydrogen carbonate (30 g). The resultant precipitate was filtered off, resuspended in ice-water, filtered off again, and dried in air and finally in vacuo over phosphorus pentaoxide; yield 90.6 g (95%), m.p. 120°-160° C, [α]D23 -33° (c 1, chloroform). in a 1-liter flask fitted with an air condenser heated in an oil bath for 3 hours at 65°-75° C The mixture was poured The resultant precipitate was filtered off filtered off again dried in air and finally in vacuo over phosphorus pentaoxide

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: A reaction mixture (100 mL, 100 mM Tris, pH 7.5, 10 mM MgCl2, 10 mM MnCl2, 5 mM EDTA, 5 ThM NaN3, 1 mm ATPe 0.01 mM theophylline and 0.03 mM 2, 3-dimercaptopropanol) containing α-methyl-D-mannopyranoside (0.4 g, 20 M), Man-1-P (about 1.5 g, about 50 mM), PEP (1.0 g, 42.7 HM), GDP (30 mg, 1 mM), dried yeast cells (0.4 g), pyruvate kinase PK (300 U), recombinant α1,2-ManT (1.2 U) and inorganic pyrophosphatase (2 U) was slightly stirred at room temperature for 72 hours, then centrifuged. The supernatant was lyophilized and extracted with methanol. After removal of the methanol, the product was purified by silica gel column chromatography (CHCl :CH3OH:H2O=6: 3:0.5, v/v/v) to afford Manα1, 2ManαOMe (Compound 3) (96 mg) in 38 percent overall yield based on consumed α-methyl-D-mannopyranoside. 1H NMR (D2O) δ3.41 (s, 3H), 3.60-3.80 (m, 6H), 3.86 (dd, 1H, J=3.5, 10 Hz), 3.88 (dd, 1H, J=3.5, 10 Hz), 3.90 (d, 1H, d, J=1.5 Hz), 3.92 (d, 1H, J=l Hz), 3.97 (dd, 1H, J=2, 3.5 Hz), 4.08 (dd, 2H, J=2, 3.5 Hz), 5.02 (d, 1H, d, J=2 Hz), 5 .04 (d, 1H, J=2.0 Hz). 13C {1H} NMR (D2O) δ54.90, 61.01, 61.24, 66.98, 67.03, 70.02, 70.29, 70.37, 72.63, 73.40, 78.62, 99.40, 102.43. HRMS calculated for C13H24O11Na+ (M+Na+) 379.1216, found 379.1220. dried yeast cells (0.4 g), pyruvate kinase PK (300 U), recombinant α1,2-ManT (1.2 U) and inorganic pyrophosphatase (2 U) extracted with methanol After removal of the methanol the product was purified by silica gel column chromatography (CHCl

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: To a stirred solution of (2R,3S,4S,5S,6S)-2-(hydroxymethyl)-6-methoxytetrahydro-2H-pyran-3,4,5-triol (15.0 g, 77.2 mmol, 1.0 equiv., commercially available Sigma Aldrich) in dry DMF (250 mL) was added sodium hydride (14.8 g, 309 mmol, 4.0 equiv) at 0° C. under nitrogen atmosphere. The reaction mixture was stirred at the same temperature for 30 minutes and then benzylbromide (38 mL, 309 mmol, 4.0 equiv) was added dropwise. The reaction mixture was stirred at room temperature for 16 hours. The reaction was quenched with MeOH (50 mL) at 0° C. and concentrated in vacuo. Ice cold water (80 mL) was added, the mixture was extracted with EtOAc (2×250 mL), and the combined organic layers were washed with saturated NaHCO3 solution (3×60 mL), dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The material thus obtained was purified by silica gel chromatography, eluting with 20% EtOAc in petroleum ether to afford (2R,3R,4S,5S,6S)-3,4,5-tris(benzyloxy)-2-((benzyloxy)methyl)-6-methoxytetrahydro-2H-pyran as a pale yellow oil (28 g, 65%). 1H NMR (400 MHz, CDCl3) δ 7.47-7.24 (m, 18H), 7.25-7.15 (m, 2H), 4.92 (d, J=10.8 Hz, 1H), 4.79 (dd, J=17.2, 2.2 Hz, 3H), 4.70 (d, J=12.1 Hz, 1H), 4.67-4.50 (m, 4H), 4.01 (t, J=9.1 Hz, 1H), 3.92 (dd, J=9.3, 3.1 Hz, 1H), 3.86-3.72 (m, 4H), 3.36 (s, 3H). ESI (M+H2O) 572.0, calc for C35H38O6 554.3. The reaction mixture was stirred at room temperature for 16 hours The reaction was quenched with MeOH (50 mL) at 0° C. concentrated in vacuo Ice cold water (80 mL) was added the mixture was extracted with EtOAc (2×250 mL) the combined organic layers were washed with saturated NaHCO3 solution (3×60 mL) dried over anhydrous sodium sulfate filtered concentrated in vacuo The material thus obtained was purified by silica gel chromatography eluting with 20% EtOAc in petroleum ether

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: A ca. 2:1 mixture of exo- and endo-isomers of bis-[(2,3-O-), (4,6-O-)]benzylidene-α-D-mannopyranoside (Carbohydrates, Ed. Collins, P. M., Chapman and Hall, New York, 1987, p. 350) was prepared by reaction of methyl α-D-mannopyranoside with 2.2 eq of α,α-dimethoxytoluene and catalytic p-toluenesulfonic acid in acetonitrile. This compound was treated with NaBH4 and HCl (Garegg, P. J.; Hultberg, H. Carbohydrate Res. 1981, 93, C10) to provide a mixture of products from which the methyl 2,6-O-benzyl-α-D-mannopyranoside was isolated by flash chromatography. The assignment of this isomer was confirmed by 1H decoupling experiments on the corresponding bis-(3,4-O-diphenylphosphino) derivative (ligand VA). 1H NMR δ7.42-7.24 (m, 10), 4.81 (d, 1, J=1 Hz), 4.75-4.54 (m, 4) 3.78-3.71 (m, 6), 3.36 (s, 3), 2.83 (bs, 1), 2.43 (bs, 1). A ca. 2:1 mixture of exo- and 1981, 93, C10) to provide was isolated by flash chromatography