Anti-adipogenic Effect of Taurine-Carbohydrate Derivatives

Experimental

Condensation of Carbohydrates with Taurine. Sodium methoxide (28% in methanol, 2.23 g, 11.6 mmol) was added to taurine (1.38 g, 11.0 mmol) in methanol (20 mL) and sonicated for a few minutes. To the resulting solution, D-glucose (1.80 g, 10.0 mmol) in methanol (20 mL) was added, sonicated for a few minutes, and stirred at 43 °C for 24 h in an oil bath. After the reaction completion, absolute ethanol (approximately 40 mL) was added dropwise at 0 °C with vigorous stirring until the precipitation stopped. The precipitate was filtered, washed with absolute ethanol, and dried in a vacuum desiccator. The reactions of other carbo-hydrates (D-galactose, D-xylose, D-arabinose, D-ribose, and D-lyxose) with taurine were also carried out in the similar manner. The reaction conditions and purification methods are listed in Table 1.

N-(β-D-Glucopyranosyl)taurine Sodium Salt (1βP): Yield: 80%; hygroscopic yellow powder, mp 76.0–91.1 °C (dec. 69.3 °C), (c = 1.0, H2O), 1H NMR (400 MHz, DMSO-d6) δ 2.61 (t, J1 = 6.4 Hz, J2 = 6.6 Hz, 2H, SCH2), 2.73–2.79 (m, 1H, NCH2), 2.84 (td, J1 = 3.2 Hz, J2 = 8.6 Hz, 1H, H-2), 3.02 (br, 2H, H-4, H-5), 3.06–3.12 (m, 2H, NCH2, H-3), 3.41–3.45 (m, 1H, H-6b), 3.62–3.66 (m, 2H, H-6a, H-1), 4.12 (q, J = 5.1 Hz, 1H, NH), 4.48 (t, J = 5.7 Hz, 1H, 6-OH), 4.65 (d, J = 3.7 Hz, 1H, 2-OH), 4.82 (br, 1H, 4-OH), 4.87 (d, J = 4.1 Hz, 1H, 3-OH); 13C NMR (100 MHz, DMSO-d6) δ 41.9 (NCH2), 51.8 (SCH2), 61.3 (C-6), 70.4 (C-5), 73.5 (C-2), 77.6 (C-3), 77.7 (C-4), 90.5 (C-1).

N-(β-D-Galactopyranosyl)taurine Sodium Salt (2βP): Yield: 80%; hygroscopic yellow powder, mp 64.8–78.0 °C (dec. 66.9 °C), (c = 1.0, H2O), 1H NMR (400 MHz, DMSO-d6) δ 2.61 (t, J1 = 7.0 Hz, J2 = 7.1 Hz, 2H, SCH2), 2.72–2.80 (m, 1H, NCH2), 3.04–3.12 (m, 1H, NCH2), 3.17 (td, J1 = 3.3 Hz, 1H, H-2), 3.23–3.28 (m, 2H, H-3, H-5), 3.41–3.46 (m, 1H, H-6b), 3.48–3.53 (m, 1H, H-6a), 3.58–3.63 (m, 2H, H-1, H-4), 4.12 (q, J = 5.2 Hz, 1H, NH), 4.27 (d, J = 4.6 Hz, 1H, 4-OH), 4.48 (d, J = 3.6 Hz, 1H, 2-OH), 4.55 (t, J = 5.5 Hz, 1H, 6-OH), 4.64 (d, J = 5.3 Hz, 1H, 3-OH); 13C NMR (100 MHz, DMSO-d6) δ 42.0 (NCH2), 52.0 (SCH2), 60.6 (C-6), 68.5 (C-4), 70.8 (C-2), 74.2 (C-3), 75.9 (C-5), 91.2 (C-1).

N-(β-D-Xylopyranosyl)taurine Sodium Salt (3βP): Yield: 84%; hygroscopic white crystal, mp 131.8–132.8 °C (dec. 99.8 °C), (c = 1.0, H2O), 1H NMR (400 MHz, DMSO-d6) δ 2.56 (t, J1 = 6.7 Hz, J2 = 6.6 Hz, 2H, SCH2), 2.65–2.68 (m, 1H, NH), 2.71–2.77 (m, 1H, NCH2), 2.82 (td, J1 = 4.1 Hz, J2 = 8.6 Hz, 1H, H-2), 2.96 (t, J = 10.8 Hz, 1H, H-5a), 2.97–3.03 (m, 1H, NCH2), 3.06 (td, J1 = 4.5 Hz, J2 = 8.7 Hz, 1H, H-3), 3.19–3.26 (m, 1H, H-4), 3.59 (t, J = 8.1 Hz, 1H, H-1), 3.63 (dd, J1 = 5.3 Hz, J2 = 11.1 Hz, 1H, H-5b), 4.61 (d, J = 4.1 Hz, 1H, 2-OH), 4.87 (d, J = 4.3 Hz, 2H, 3-OH, 4-OH); 13C NMR (100 MHz, DMSO-d6) δ 41.9 (NCH2), 51.9 (SCH2), 66.7 (C-5), 69.9 (C-4), 73.4 (C-2), 77.4 (C-3), 91.5 (C-1).

N-(β-D-Arabinopyranosyl)taurine Sodium Salt (4βP): Yield: 72%; hygroscopic white crystal, mp 114.9–115.7 °C (dec. 96 °C), (c = 1.0, H2O), 1H NMR (400 MHz, DMSO-d6) δ 2.62 (t, J1 = 6.4 Hz, J2 = 6.3 Hz, 2H, SCH2), 2.73–2.78 (m, 2H, NCH2, NH), 3.01–3.07 (m, 1H, NCH2), 3.24 (td, J1 = 4.0 Hz, J2 = 8.0 Hz, 1H, H-3), 3.32–3.35 (m, 2H, H-2, H-5), 3.62–3.65 (m, 3H, H-1, H-4, H-5), 4.48 (d, J = 3.9 Hz, 1H, 4-OH), 4.57 (d, J = 4.0 Hz, 1H, 3- OH), 4.71 (d, J = 5.2 Hz, 1H, 2-OH); 13C NMR (100 MHz, DMSO-d6) δ 42.0 (NCH2), 52.0 (SCH2), 65.6 (C-5), 67.9 (C-4), 70.8 (C-3), 73.2 (C-2), 91.0 (C-1).

N-(D-Ribopyranosyl)taurine Sodium Salt (5αP, 5βP): Yield: 82%; hygroscopic white powder, mp 73.6–84.5 °C (dec. 66 °C), (c = 1.0, H2O), 1H NMR (400 MHz, D2O): δ ppm 3.00–3.09 (m, 3.5H), 3.10–3.15 (m, 5.5H), 3.17–3.25 (m, 1.5H), 3.28–3.34 (m, 1.5H), 3.43 (dd, J1 = 2.9 Hz, J2 = 8.4 Hz, 1H), 3.61 (dd, J1 = 1.3 Hz, J2 = 12.9 Hz, 1.5H), 3.62 (q, J = 10.8 Hz, 1.5H), 3.72 (dd, J1 = 4.9 Hz, J2 = 10.9 Hz, 1H), 3.83–3.85 (m, 1.5H), 3.89 (m, 1.5H), 3.95 (dd, J1 = 2.9 Hz, J2 = 12.8 Hz, 1.5H), 4.13 (t, J = 2.8 Hz, 1H), 4.16 (s, 1.5H), 4.24 (d, J = 8.4 Hz, 1H); 13C NMR (100 MHz, D2O) δ 40.4, 40.7, 50.9, 51.0, 63.2, 66.8, 68.3, 68.6, 70.0, 70.1, 71.2, 86.6, 87.5.

N-(D-Lyxopyranosyl)taurine sodium salt (6αP, 6βP): Yield: 78%; hygroscopic white powder, mp 77.8–89.2 ˚C (dec. 70.8 ˚C), (c = 1.0, H2O), 1H NMR (400 MHz, D2O) δ 2.97–3.04 (m, 2.5H), 3.06–3.11 (m, 4H), 3.13–3.28 (m, 3.5H), 3.55 (dd, J1 = 3.4 Hz, J2 = 9.6 Hz, 1H), 3.60–3.64 (m, 1H), 3.67 (dd, J1 = 3.3 Hz, J2 = 7.1 Hz, 1H), 3.75 (dd, J1 = 5.4 Hz, J2 = 9.8 Hz, 1H), 3.78–3.82 (m, 2H), 3.87–3.89 (m, 1H), 3.91 (d, J = 5.3 Hz, 1H), 4.17 (s, 1H), 4.28 (d, J = 7.0 Hz, 1H); 13C NMR (100 MHz, D2O) δ 40.5, 50.8, 50.8, 64.2, 66.2, 66.6, 68.6, 70.4, 70.7, 73.7, 87.2, 87.8.

Human Preadipocytes Culture and Differentiation into Adipocytes. Human preadipocytes were seeded into 6-well plates (1.8 × 105 cell per well in 2 mL of the medium) and cultured in 5% carbon dioxide at 37 °C until confluent. In order to make the preadipocytes differentiate into adipocytes, the culture medium was changed to an adipocyte differentia-tion medium, and the preadipocytes were cultured for two weeks by changing the medium every three days in the presence of taurine-carbohydrate derivatives at different concentrations (0-100 µg/mL).

Oil Red O Staining and Measurement of Optical Density Value. After the removal of the culture solution, the cultured cells were washed twice with phosphate-buffered saline and kept in 100 mL/L formaldehyde solution for 1 h. Then, the formaldehyde solution was removed, and the cells were rinsed twice with deionized water. Next, the cells were stained with oil red O solution (60% in isopropanol) for 20 min at room temperature. After removing the staining solu-tion, the cultured cells were observed using an optical micro-scope and pictures were recorded. Next, the dye retained in the cells was eluted with isopropanol, and the OD values were measured at the optical absorbance of 500 nm using an Emax microplate reader.