*4.4. Purification and Identification of the Glycosylated Product*

Twenty-five mL of the reaction mixture (1 mg/mL GAA, 15 μg/mL BsGT110, 10 mM UDP-glucose, 10 mM MgCl2, 50 mM acetate buffer at pH 6) was carried out at 40 ◦C for 30 min. Afterward, an equal volume of methanol was added into the reaction mixture to stop the reaction. Fifty mL of the reaction mixture with 50% methanol was applied to a preparative YL9100 HPLC system (YoungLin, Gyeonggi-do, Korea). The stationary phase was the Inertsil ODS 3 column (10 mm, 20 i.d. × 250 mm, GL Sciences, Eindhoven, The Netherlands), and the mobile phase was the same as that in the UPLC system, but with a flow rate of 15 mL/min. The detection condition was 254 nm, and the sample volume was 10 mL for each injection. The product of each run was collected, concentrated under a vacuum, and lyophilized with a freeze dryer. From the 25 mL of reaction, 5.4 mg of the product was purified. The chemical structure of the product compound was determined with mass and NMR spectral analyses. The mass spectral analysis was performed on a Finnigan LCQ Duo mass spectrometer (ThermoQuest Corp., San Jose, CA, USA) with ESI. 1H- and 13C-NMR, HSQC, and HMBC spectra were recorded on a Bruker AV-600 NMR spectrometer (Bruker Corp., Billerica, MA, USA) at ambient temperature. Standard pulse sequences and parameters were used for the NMR experiments, and all chemical shifts were reported in parts per million (ppm, δ).
