Diaion hp20 column

EC, ECG, EGC, EGCG, TF, TF3G, TF3′G, and TFDG were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). TSA was synthesized from EGCG and purified as described [26 (link)]. Briefly, a solution of EGCG (1.0 g, 2.18 mmol) and CuCl₂ (269 mg, 2.00 mmol) in 30% MeOH (400 mL) was vigorously mixed for 24 h. To the reaction mixture, ascorbic acid (10 g) was added and heated at 85 °C for 15 min. After chilling, the mixture was concentrated to evaporate MeOH and the resulting aqueous solution was applied to a Diaion HP20 column (Mitsubishi Chemical Corp., Tokyo, Japan, 3.0 cm i.d. × 25 cm) with water. After washing the column with water to remove reagents, sample was eluted with 70% MeOH (300 mL), and applied to a preparative high-performance liquid chromatography (HPLC) using a YMC-Actus Triart C18 column (YMC Co., Ltd., Kyoto, Japan, 20 mm i.d. × 250 mm). To elute the column, isocratic elution method was performed using a solvent mixture of water: CH₃CN = 3:2 at a flow rate of 16 mL/min. LC chromatograms were obtained at UV 280 nm. The fraction of TSA was collected and freeze-dried. The yield and purity of TSA were 251 mg and 96%, respectively.

Trp-Leu, Leu-Trp, Trp-Met, Trp-Ala, Trp-Tyr, and Trp-Asp were purchased from Bachem AG (Bubendorf, Switzerland). EGCG was purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan). TSA and theasinensin B (TSB) were prepared as described in our previous report [24 (link)]. Briefly, a solution of EGCG incubated with CuCl₂ and ascorbic acid was applied to a Diaion HP20 column (Mitsubishi Chemical Co., Tokyo, Japan), followed by a stepwise elution with methanol to obtain TSA [24 (link)]. TSB was obtained from a fermented green tea product [25 (link)]. An internal standard (IS), 3-trimethylsilyl-1-propanesulfonic acid-d₆ (DSS-d₆, 98.0 atom% D) was obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, TX, USA). Deuterium oxide (D₂O, 99.8 atom% D) was obtained from Acros Organics (Fair Lawn, NJ, USA). Other reagents were of analytical grade and were used without further purification.
Log P value of each dipeptide was obtained by a SciFinder Substance Identifier software (https://scifinder.cas.org/scifinder/view/scifinder/scifinderExplore.jsf).