GALACTOSE

反应条件: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

实验步骤: α-Gal A was assayed using 4-methylumbelliferyl-α-D-galactopyranoside (4-MU-α-Gal) as previously described (Bishop, et al., 1980, In Enzyme Therapy in Genetic Diseases: 2. Desnick, R. J. (Ed.). Alan R. Liss, Inc. New York, p. 17). Briefly, a stock solution of 5 mM 4-MU was prepared in 0.1M citrate-phosphate buffer, pH 4.6 solubilized in an ultrasonic bath. The reaction mixtures containing 10-50 μl of enzyme preparation or cell extracts and 150 μl substrate, were incubated at 37° C. for 10-30 min. The reactions were terminated with the addition of 2.33 ml of 0.1M ethylenediamine. One unit of activity is that amount of enzyme which hydrolyzes 1 nmol of substrate/hour. pH 4.6 solubilized in an ultrasonic bath The reaction mixtures containing 10-50 μl of enzyme preparation or cell extracts and 150 μl substrate The reactions were terminated with the addition of 2.33 ml of 0.1M ethylenediamine

反应条件: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

实验步骤: AmB (800 mg, 0.86 mmol) was dissolved in 16 mL anhydrous dimethylformamide (DMF) and stirred for 5 min. α-D-Glucose (316 mg, 1.72 mmol) was added. The resulting solution was sealed to the atmosphere and placed in a water bath equilibrated to 37° C. for 6 hr. The reaction was followed by LCMS until judged complete. The reaction product was used directly in the amidation reaction, described infra, without purification. The corresponding D-galactose, D-maltose, D-cellobiose and α-D-galactose glycosylated rearrangement products were obtained using the same procedure, but substituting D-galactose (315 mg, 1.72 mmol), D-maltose hydrate (620 mg, 1.72 mmol), D-cellobiose (588 mg, 1.72 mmol) and α-D-lactose hydrate (620 mg, 1.72 mmol), respectively, for α-D-glucose. was added The resulting solution was sealed to the atmosphere The reaction product was used directly in the amidation reaction without purification

反应条件: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

实验步骤: AmB (800 mg, 0.86 mmol) was dissolved in 16 mL anhydrous dimethylformamide (DMF) and stirred for 5 min. α-D-Glucose (316 mg, 1.72 mmol) was added. The resulting solution was sealed to the atmosphere and placed in a water bath equilibrated to 37° C. for 6 hr. The reaction was followed by LCMS until judged complete. The reaction product was used directly in the amidation reaction, described infra, without purification. The corresponding D-galactose, D-maltose, D-cellobiose and α-D-galactose glycosylated rearrangement products were obtained using the same procedure, but substituting D-galactose (315 mg, 1.72 mmol), D-maltose hydrate (620 mg, 1.72 mmol), D-cellobiose (588 mg, 1.72 mmol) and α-D-lactose hydrate (620 mg, 1.72 mmol), respectively, for α-D-glucose. was added The resulting solution was sealed to the atmosphere The reaction product was used directly in the amidation reaction without purification

反应条件: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

实验步骤: AmB (800 mg, 0.86 mmol) was dissolved in 16 mL anhydrous dimethylformamide (DMF) and stirred for 5 min. α-D-Glucose (316 mg, 1.72 mmol) was added. The resulting solution was sealed to the atmosphere and placed in a water bath equilibrated to 37° C. for 6 hr. The reaction was followed by LCMS until judged complete. The reaction product was used directly in the amidation reaction, described infra, without purification. The corresponding D-galactose, D-maltose, D-cellobiose and α-D-galactose glycosylated rearrangement products were obtained using the same procedure, but substituting D-galactose (315 mg, 1.72 mmol), D-maltose hydrate (620 mg, 1.72 mmol), D-cellobiose (588 mg, 1.72 mmol) and α-D-lactose hydrate (620 mg, 1.72 mmol), respectively, for α-D-glucose. was added The resulting solution was sealed to the atmosphere The reaction product was used directly in the amidation reaction without purification

反应条件: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

实验步骤: A mixture of p-aminophenyl 3-O-α-D-galactopyranosyl-α-D-galactopyranoside 22 (11 mg, 0.025 mmol), tetrameric platform 28, and NaHCO3 (3 mg, 0.030 mmol) in 0.15 mL of H2O/CH3CN (1:1) was slightly shaken for 1 h. After addition of 0.15 mL of H2O, the reaction mixture was set at room temperature for 2 d and purified on reversed phase HPLC column eluted at 1 mL/min with a gradient of acetonitrile-water (0 to 30%) over 15 minutes to yield 41 (4.6 mg, 40%) as a white solid: MS (ESI): m/e (M/2+1) Calcd. for C60H95N7O29: 1377.6 obsd: 1377.9. the reaction mixture was set at room temperature for 2 d purified on reversed phase HPLC column eluted at 1 mL/min with a gradient of acetonitrile-water (0 to 30%) over 15 minutes

反应条件: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

实验步骤: A reaction scheme illustrating the chemoenzymatic synthesis of the αGal epitope, p-aminophenyl 3-O-β-D-galactopyranosyl-α-D-galactopyranoside (22) is shown in FIG. 3. p-Nitrophenyl 3-O-α-D-galactopyranosyl-α-D-galactopyranoside (compound 21) was prepared enzymatically as described by Nilsson (Tetrahedron Lett. (1997) 38:133-136). A mixture of p-nitrophenyl 3-O-β-galactopyranosyl-α-D-galactopyranoside (70 mg, 0.15 mmol) and 10 mg Pd/C (10%) in methanol (4 mL) was stirred under hydrogen at room temperature overnight. The reaction mixture was then filtered through Celite and the filtrate was concentrated in vacuo to give (50 mg) as a yellowish solid: 1H NMR (CD3OD): δ 6.87 (t, 2 H), 6.57 (d, 2 H), 5.21 (d, 1 H), 4.98 (d, 1 H), 4.2-4.1 (m, 2 H), 4.1-3.8 (m, 3 H), 3.8-3.7 (m, 3 H), 3.7-3.5 (m,4 H); 13C NMR (CD3OD): δ 143.8, 120.1, 117.9, 115.4, 101.2, 97.6, 78.3, 77.0, 72.6, 71.5, 71.4, 70.3, 68.7, 67.4, 63.2, 62.6; MS (ESI): m/e (M+Na+) Calcd. for C18H27NO11Na: 456.2, obsd: 456.2. A reaction scheme The reaction mixture was then filtered through Celite the filtrate was concentrated in vacuo to give (50 mg) as a yellowish solid

反应条件: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

实验步骤: α-Gal A was assayed using 4-methylumbelliferyl-α-D-galactopyranoside (4-MU-α-Gal) as previously described (Bishop, et al., 1980, In Enzyme Therapy in Genetic Diseases: 2. Desnick, R. J. (Ed.). Alan R. Liss, Inc. New York, p. 17). Briefly, a stock solution of 5 mM 4-MU was prepared in 0.1M citrate-phosphate buffer, pH 4.6 solubilized in an ultrasonic bath. The reaction mixtures containing 10-50 μl of enzyme preparation or cell extracts and 150 μl substrate, were incubated at 37° C. for 10-30 minutes. The reactions were terminated with the addition of 2.33 ml of 0.1M ethylenediamine. One unit of activity is that amount of enzyme which hydrolyzes 1 nmol of substrate/hour. pH 4.6 solubilized in an ultrasonic bath The reaction mixtures containing 10-50 μl of enzyme preparation or cell extracts and 150 μl substrate The reactions were terminated with the addition of 2.33 ml of 0.1M ethylenediamine

反应条件: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

实验步骤: α-Gal A was assayed using 4-methylumbelliferyl-α-D-galactopyranoside (4-MU-α-Gal) as previously described (Bishop, et al., 1980, In Enzyme Therapy in Genetic Diseases:2. Desnick, R. J. (Ed.). Alan R. Liss, Inc. New York, p. 17). Briefly, a stock solution of 5 mM 4-MU was prepared in 0.1M citratephosphate buffer, pH 4.6 solubilized in an ultrasonic bath. The reaction mixtures containing 10-50 μl of enzyme preparation or cell extracts and 150 μl substrate, were incubated at 37° C. for 10-30 min. The reactions were terminated with the addition of 2.33 ml of 0.1M ethylenediamine. One unit of activity is that amount of enzyme which hydrolyzes 1 nmol of substrate/hour. pH 4.6 solubilized in an ultrasonic bath The reaction mixtures containing 10-50 μl of enzyme preparation or cell extracts and 150 μl substrate The reactions were terminated with the addition of 2.33 ml of 0.1M ethylenediamine

反应条件: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

实验步骤: α-D-galactopyranose; β-D-galactopyranose; α-D-galactofuranose; β-D-galactofuranose; L-galactose; α-L-galactopyranose; β-L-galactopyranose; α-L-galactofuranose; β-L-galactofuranose

反应条件: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

实验步骤: α-Gal A was assayed using 4-methylumbelliferyl-α-D-galactopyranoside (4-MU-α-Gal) as previously described (Bishop, et al., 1980, In Enzyme Therapy in Genetic Diseases: 2. Desnick, R. J. (Ed.). Alan R. Liss, Inc. New York, p. 17). Briefly, a stock solution of 5 mM 4-MU was prepared in 0.1M citrate-phosphate buffer, pH 4.6 solubilized in an ultrasonic bath. The reaction mixtures containing 10-50 μl of enzyme preparation or cell extracts and 150 μl substrate, were incubated at 37° C. for 10-30 min. The reactions were terminated with the addition of 2.33 ml of 0.1M ethylenediamine. One unit of activity is that amount of enzyme which hydrolyzes 1 nmol of substrate/hour. pH 4.6 solubilized in an ultrasonic bath The reaction mixtures containing 10-50 μl of enzyme preparation or cell extracts and 150 μl substrate The reactions were terminated with the addition of 2.33 ml of 0.1M ethylenediamine