C18 column

Metabolite extraction and antimicrobial activity analysis for LC-MS was conducted as previously described by Choi et al. (2008 (link)). The final methanol extract was evaporated and dissolved in 2 ml water. The concentrated samples were further purified on a C18 column (Sili-Cycle Inc. Quebeck, CA) and eluted with a water/methanol gradient. Active eluted fractions were used for LC-MS. LC-MS was performed using a high pressure liquid chromatography system (Thermo Electron Co, USA). The samples were injected into reverse phase columns and analyzed in a mixed solvent of water and acetonitrile containing 0.1% formic acid (0.2 ml/min).
Lipopeptide extraction for MALDI-TOF MS was performed as previously described by Li et al. (2007 (link)). Bruker Ultraflex II mass spectrometer using alpha-hydroxycinnamic acid (alpha-HCCA) was used as matrix and spectra were visualized with the mMass software (Niedermeyer and Strohalm, 2012 (link)).

A mixture of CUR (368 mg, 1 mmol), MPA (320 mg, 1 mmol), and DMAP (24 mg, 0.2 mmol) was dissolved in acetone (10 mL) to get a clear solution. The mixture was then added dropwise with a solution of EDC (287.55 mg, 1.5 mmol) in acetone (5 mL). The reaction mixture was stirred on an ice bath and kept at the temperature range between 0 and 5 °C for 30 min. To the reaction, 0.1 N HCl (10 mL) was added and stirred for 1 min. The reaction mixture was extracted with dichloromethane (3 × 30 mL). The combined dichloromethane extract was washed with DI water (2 × 20 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure (Laborota 4003, Heidolph, Schwabach, Germany) to obtain a crude product in yellow (107 mg, 16% yield). The crude product was subjected to column chromatographic purification on a C18 column (40–63 µ, SiliCycle Inc., Quebec City, Quebec, Canada) using a mixture of methanol and water (8:2) as a mobile phase. Each eluent fraction was monitored by thin-layer chromatography (TLC) on a silica gel 60 F₂₅₄ plate (0.25 mm thickness) using a mixture of dichloromethane and methanol (15:1) as a developing solvent.