Rp c18

The wild-type and ppGpp⁰ strains were cultured in the GYM medium with 2% inoculum size, and the samples were taken at intervals of 12 h. The production of toyocamycin and tetraene macrolides was analyzed by the high-performance liquid chromatograph (HPLC) method. The toyocamycin was performed on a Waters 2950 HPLC system, including a PDA detector. Then, 10 μL of the samples were injected into a column (RP-C18, 250 mm × 4.6 mm, 5 μm, Waters, USA) with a water-methanol gradient system with a flow rate of 1 mL/min. The methanol ranged linearly from 5% to 100% over 30 min and was then held for 10 min. The detection wavelength was 279 nm. The column temperature was maintained at 30°C.
The tetramycin P was determined using the RP-C18 (250 mm × 4.6 mm, 5 μm, Waters, USA) column, with the water-methanol gradient system, with a detection wavelength of 304 nm and a solvent flow rate of 1.0 mL/min. The gradient system ranged linearly from 15% to 100% methanol over 20 min and was then held for 8 min. The injection volume was 5 μL. The column temperature was maintained at 30°C.

Optical rotations were measured on a JASCO P-1020 digital polarimeter (Jasco Corp., Tokyo, Japan). UV spectra were recorded on a Thermo Scientific Multiskan GO microplate spectrophotometer (Thermo Scientific Co., Waltham, MA, USA) in MeOH. ECD spectra were recorded on a JASCO J-815 circular dichroism spectrometer (JASCO Electric Co., Ltd., Tokyo, Japan). IR spectra were determined on a Nicolet-Nexus-470 spectrometer (Thermo Electron Co., Madison, WI, USA) using KBr pellets. 1D and 2D NMR spectroscopic data (TMS as an internal standard) were acquired on Agilent DD2-500 (JEOL, Tokyo, Japan) and Bruker AV-600 NMR spectrometer (Bruker BioSpin GmbH Co., Rheinstetten, Germany). ESIMS and HRESIMS spectra were obtained from a Micromass Q-TOF spectrometer (Waters Corp., Manchester, UK), and a Thermo Scientific LTQ Orbitrap XL spectrometer (Thermo Fisher Scientific, Bremen, Germany). Semipreparative HPLC was performed on a Shimadzu LC-20 AT system using a RP-C18 (Waters, 5 μm, 10 × 250 mm) column.