反应详情

EC (1 g, 3.5 mmol) and EGC (1 g, 3.3 mmol) were dissolved into the 200 mL of phosphate-citrate buffer (50 mM, pH 5.0) along with 2 g of crude PPO enzyme. The enzymatic oxidation was carried out at room temperature for 6 hour with stirring. The reaction solution was then subjected to fractionation with the same volume of ethyl acetate with three times. Then, the organic layer was concentrated under reduced pressure. The resulting residues were subjected to Sephadex LH-20 column chromatography eluting with gradient of ethanol to 20% of acetone in ethanol. Among the collected 14 fractions (each c.a. 90 mL), 8˜10 fractions were combined, and concentrated under reduced pressure. The resulting residue was subjected to further purification on a RP-18 silica gel column eluting with gradient of 40%˜50% of aqueous methanol. During elution, 38 fractions (each c.a. 13 mL) were received. Among them, 10˜17 fractions were combined, and concentrated under reduced pressure, and were subjected to freeze-drying. It yielded deep-reddish color of compound 1 (280 mg). Along with the same enzyme reaction and isolation procedure, compound 2 was obtained from EC and EGCG reaction. The enzymatic oxidation of EGC and ECG, ECG and EGCG reaction yielded compound 3 and compound 4, respectively.