Scaled-up Compound Purification and NMR Structural Elucidation

To obtain sufficient compound for structural elucidation by NMR, cultures were scaled up to 500 mL batches to a total volume of 5 Liters and expressed using the combinatorial expression system described above (Kitaoka et al., 2015 (link)). The resultant products were extracted with an equal volume of hexanes. The organic extract was recovered using a separatory funnel and then dried by rotary evaporation. The resulting residue was passed through a silica column and eluted using a hexane: ethyl acetate gradient. Fractions of interest were further purified by HPLC using an Agilent 1260 series instrument equipped with an autosampler, fraction collector and diode array UV detector over a ZORBAX Eclipse XDB C18 (4.6 x 150 mm, 5 mm) at a 0.5 mL/min flow rate using a water and acetonitrile gradient as the mobile phase. Purified compounds were resuspended in CDCl₃. Structural analysis was performed using 1D (^‘1H, 13C^’) and 2D (TOCSY, ROESYPHPR, HMBCGP, DQFCOSY, and 13CHSQC) NMR experiments. Spectra were acquired on a Bruker Avance 500-MHz TopSpin NMR spectrometer.