65.4% |
With Escherichia coli thymidine phosphorylase; In aq. phosphate buffer; at 40℃;pH 6.8;Enzymatic reaction;Kinetics; |
General procedure: Thymidine phosphorylase (from Escherichia coli, EC 2.4.2.4) was purchased from Sigma-Aldrich Chemical Co. The units of enzymatic activity indicate the transition of native substrates as 1.0 mumol each of thymidine and phosphate to thymine and 2-deoxyribose-1-phophate per 1 min. In this research, a ?unit? refers to the amount of enzyme activity, even though both synthetic and natural substrates were in use. Incubations generally contained 40 mM thymidine (0.4 mmol, 0.097 g), 5 mM various uracil derivatives (incorporated stable isotopes, 0.05 mmol) and 1 unit/mL of thymidine phosphorylase in 10 mL of 1 mM phosphate buffer (pH 6.8). Mixtures were stirred at 40 C until the reactions reached equilibrium. Product formation was monitored via UV absorption at 254 nm using HPLC with a C18 column (4.6 mm diameter, 5% MeCN in 1 mM phosphate buffer; pH6.8). After removal of H2O in vacuo, the residue was purified using preparative HPLC (C-18 reverse phase column, 4.6 mm diameter, Unison UK-C18, Imtakt Co., Kyoto) by eluting with an H2O-MeCN system (as above) to afford a pure nucleoside incorporating stable isotopes. 4.2.2 1,3-15N2-uridine 1H NMR (CD3OD) delta 7.97 (1H, J = 8.4 Hz, d), 6.26 (1H, J = 6.6 Hz, t), 5.69 (1H, m), 4.38 (1H, m), 3.92 (1H, m), 3.73 (2H, m), 2.23 (1H, m). 13C NMR (CD3OD) delta 166.3 (J = 9.5 Hz, d), 152.5 (J = 18, 18 Hz, dd), 142.7 (J = 12 Hz, d), 102.6 (J = 6.7 Hz, d), 90.6 (J = 11 Hz, d), 86.3, 82.5, 75.7, 71.2, 62.2. EIMS m/e 247 [M]+. |