Reaction Steps
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.
