4-Nitrophenyl-beta-D-galactopyranoside synthesis

Yield:-

Reaction Conditions:

Stage #1: D-Galactosewith pyridine at 70; for 1 h;
Stage #2: acetic anhydride at 23 - 70; for 25 h;
Stage #3: 4-nitro-phenolOverall yield = 1.32 g; Further stages;

Steps:

3.2 Isomerization and acetylation to obtain per-acetylated galactofuranose (4)

General procedure: To 9 pyridine (30mL) was added d-galactose (1.81g, 10.0mmol) while stirring at 70°C. After 1h, acetic anhydride (6.00mL, 63.5mmol) was added into a reaction mixture at 70°C and the mixture was stirred for 1h. Then the reaction mixture was cooled to 23°C, stirred for 24h, and then concentrated in vacuo. The resulting syrup was diluted with EtOAc (100mL), washed with water (100mL), 1m aq. HCl (100mL), and brine, followed by drying over MgSO₄. Concentration in vacuo gave a mixture of per-acetylated galactofuranose (4) and per-acetylated galactopyranose (4p) in 96% yield (3.75g, 9.60mmol). ¹H NMR (CDCl₃): δ5.73-5.70 (m, 0.6H, H-1_4p), 5.43-5.41 (m, 0.4H, H-1₄), 5.35-5.28 (m, 1H, H-3), 5.08-5.02 (m, 1H, H-2), 4.37-4.31 (m, 1H, H-4), 4.18-4.16 (m, 1H, H-5), 4.13-4.08 (m, 2H, H-6), 2.16 (s, 3H, Ac), 2.12 (s, 3H, Ac), 2.10 (s, 3H, Ac), 2.04 (s, 3H, Ac), 1.99 (s, 3H, Ac); ¹³C NMR (CDCl₃): δ 170.3, 170.1, 169.9, 169.8, 169.4, 169.0, 99.1, 93.0, 92.1, 89.7, 82.1, 80.6, 79.0, 76.3, 75.3, 73.3, 71.7, 70.8, 70.3, 69.2, 68.7, 67.8, 67.4, 67.3, 66.8, 66.4, 62.5, 62.1, 61.2, 61.0, 21.0, 20.9, 20.8, 20.7, 20.6, 20.5, 20.4; Anal. Calc. for: C₁₆H₂₂O₁₁: C, 49.23; H, 5.68. Found: C, 49.28; H, 5.87 To a solution of 4 and 4p (2:3, 3.72g, 9.55mmol) with MS4A in CH₃CN (50mL) was added p-nitrophenol (3.99g, 28.7mmol) and BF₃·Et₂O (3.63mL, 28.67mmol) at 0°C. The reaction mixture was stirred at the same temperature for 1h and then at 23°C for 24h. Next the mixture was filtered through a Celite pad and the filtrate was concentrated in vacuo. The residue was diluted with EtOAc (100mL), washed with sat. aq. NaHCO₃ solution (100mL x 8) to remove excess p-nitrophenol as a sodium salt and brine (50mL), dried over MgSO₄, and concentrated in vacuo to dryness, producing a mixture of 42 p-nitrophenyl 2,3,4,6-tetra-O-acetyl-β-d-galactofuranoside (5) and 13 p-nitrophenyl 2,3,4,6-tetra-O-acetyl-β-d-galactopyranoside (5p) as a brown syrup (3.77g, 8.02mmol, 84%). The mixture of 5 and 5p was used for deprotection and hydrolysis without any purification^{. 1}H NMR (CDCl₃): δ 8.21 (m, 2H, J_o,m=9.2Hz, Ph(m)), 7.19 (d, 2H, J_o,m=9.2Hz, Ph(o)), 5.88 (d, 1H, J_1,2=4.0Hz, H-1), 5.57-5.54 (m, 1H, H-4), 5.53-5.52 (m, 1H, H-5), 5.31 (dd, 1H, J_2,3=4.0Hz, J_3,4=10.8Hz, H-3), 4.25 (t, 1H, J_1,2=J_2,3=4.0Hz, H-2), 4.13-4.03 (m, 2H, H-6), 2.18 (s, 3H, Ac), 2.08 (s, 3H, Ac), 2.04 (s, 3H, Ac), 1.93 (s, 3H, Ac); ¹³C NMR (CDCl₃): δ 170.4, 170.3, 170.2, 170.1, 170.0, 169.9, 169.6, 169.3, 160.7, 142.8, 126.2, 125.8, 116.5, 103.7, 98.6, 94.7, 89.7, 81.8, 81.2, 76.2, 69.1, 68.7, 67.8, 67.2, 66.4, 62.4, 61.3, 61.2, 20.9, 20.8, 20.7, 20.6, 20.5; Anal. Calc. for: C₂₀H₂₃NO₁₂+H₂O: C, 47.81; H, 4.81; N, 2.79. Found: C, 47.59; H, 4.89; N, 2.67. To a solution of 5 and 5p (2.07 g, 4.41 mmol) in CH3OH (10 mL)was added 28% aq. NH3 solution (10 mL) at 0 °C. The resulting solutionwas stirred at the same temperature for 1 h and then at 23 °C for 24 h.The reaction solution was concentrated using a rotary evaporator(35 °C), and the resulting syrup was crystalized with Et2O to afford themixture of p-nitrophenyl β-D-galactofuranoside (1) and p-nitrophenyl β-D-galactopyranoside (1p) as a brown solid (1.32 g, 4.39 mmol, 99%).To a solution of the above mixture of 1 and 1p (0.58 g, 1.9 mmol) in50mM phosphate buffer (pH 7.2, 20 mL) was added β-galactosidase fromEscherichia coli (FUJIFILM Wako Pure Chemical Corporation,>600Units/mg, 1.0 mg) followed by stirring at 37 °C for 24 h. After confirmingselective pyranoside hydrolysis with TLC analysis, the reaction mixturewas washed with CHCl3 (20 mL x 3), and then concentrated in vacuo.Target material was purified by silica-gel column chromatography(CHCl3:CH3OH, 4/1) to give p-nitrophenyl β-D-galactofuranoside (pNPGalf,1) as a colorless solid (0.25 g, 0.83 mmol, 43%).

References:

Ota, Ryo;Okamoto, Yumi;Vavricka, Christopher J.;Oka, Takuji;Matsunaga, Emiko;Takegawa, Kaoru;Kiyota, Hiromasa;Izumi, Minoru [Carbohydrate Research,2019,vol. 473,p. 99 - 103]