Isco combiflash rf 200

Large scale shake flask culture was utilized for compound isolation. A starter culture of S1 transformed with pJB034 was prepared by stabbing through a patch and inoculating 2 mL of liquid uracil dropout media in a culture tube. This starter culture was shaken at 28 °C and 250 rpm for 24 hours and then transferred to 50 mL liquid uracil dropout. After 24 hours, 2 L culture was inoculated. 24 hours later 340 mg 8-hydroxygeraniol was fed to 2 L culture to a final concentration of 1 mM. After 24 hours, the culture was spun down. The supernatant was extracted with an equal volume of ethyl acetate and the pellet was extracted with an equal volume of 50:50 mixture of ethyl acetate:acetone. The organic fractions were combined and dried. The resulting crude extract was separated using ISCO-CombiFlash Rf 200 (Teledyne Isco) with a gradient of hexane and acetone. The fractions containing the reduced shunt product were pooled and purified with HPLC using a Shimadzu Prominence HPLC (Phenonenex Kinetex, 5μ, 10.0 × 250 mm, C-18 column). The elution method was a linear gradient of 35–55% (v/v) CH₃CN/H₂O in 20.5 minutes, followed by a linear gradient of 55–95% in 2 minutes, followed by 95% CH₃CN/H₂O for 3 minutes with a flow rate of 3.0 mL/min. Structures were solved by NMR (Fig. S13 – S22, Table S3 – S4).

From yeast, the ethyl acetate extract from a 4 L YPD liquid medium was evaporated to dryness to yield the crude extract. From P. herquei, the acetone extract from a 2 L PDA solid agar extract of mutant was evaporated to dryness and partitioned between ethyl acetate/H₂O three times. To purify the desired compound, crude extracts were separated by Sephadex-LH20, reverse phase-C18 and additional HPLC steps as required. Generally, the crude extract obtained from the yeast culture or P. herquei cultivated on the PDA solid agar plates was submitted to Sephadex-LH20 eluted with methanol. After analysis by the LC-MS, the fractions containing the target compound were combined and then submitted to separation by ISCO-CombiFlash® Rf 200 (Teledyne Isco, Inc) with a reverse phase-C18 column eluted by a gradient of methanol and water. After analysis by LC-MS, the fractions containing the target compound was combined and further purified by semi-preparative HPLC using C18 reverse-phase column. The purity of each compound was checked by LC-MS, and the structure was confirmed by NMR. ¹H, ¹³C and 2D NMR spectra were obtained using DMSO-d₆ or CD₃OD as solvent on Bruker AV500 spectrometer with a 5 mm dual cryoprobe at the UCLA Molecular Instrumentation Center.