Lc 20ap

PCA (>95%), 2-OH-PCA (99.4% purity) and 2-OH-PHZ (99.0% purity) were prepared by our laboratory (Laboratory of Microbial Resources and Metabolic Engineering). The red pigment precipitate from the GP72 culture was suspended in water, and extracted using a double volume of acetic acid/ether, then evaporated by rotary evaporation. Further purification was performed by preparative liquid chromatography (Shimadzu LC-20AP) using a previously described method [11] (link). 2-OH-PCA (99.4%purity) was prepared and used for the following experiments. HPLC grade methanol (Lingfeng Chemical Reagent Co. Ltd., Shanghai, China) and ammonium acetate (Sigma Chemicals Co.) were used for the HPLC analysis, and all other chemicals were reagent grade (Sinopharm Chemical Reagent Co. Ltd., Shanghai, China). The pure water was produced by a water purifier (Aquapro DZG-303A; Zhongqin, China).

All chemicals were obtained from commercial suppliers (Adamas and Alfa), and used without further purification, unless otherwise indicated. VHL-1 based acid and cis VHL-1 based acid were prepared according to the ref. 97 . HPLC preparation was performed on SHIMADZU LC-20AP instrument with original column. All new compounds were characterized by 1H NMR and HRMS. 1H NMR spectra were recorded on Bruker AVANCE III 500 MHZ (operating at 500 MHz for 1H NMR), chemical shifts were reported in ppm relative to the residual CDCl3 (7.26 ppm 1H), DMSO-d6 (2.50 ppm 1H) or Methanol-d4 (3.31 ppm, 1H), and coupling constants (J) are given in Hz. Multiplicities of signals are described as follows: s --- singlet, d --- doublet, t --- triplet, m --- multiple. High Resolution Mass Spectra were recorded on AB Triple 4600 spectrometer with acetonitrile and water as solvent.

Crude lyophilized conjugates
were dissolved in either 22% MeCN/H₂O (P3 and P4) or 22%
MeCN/25% DMF/H₂O (P1, P2, P5, and P6) and purified by semipreparative
HPLC on a LC20-AP instrument (Shimadzu, Kyoto, Japan) using Gemini
C₁₈ column (10 μm, 110 Å, 10 × 250 mm,
Phenomenex) and a linear 15%–95% MeCN gradient into 0.1% TFA
in H₂O over 40 min at 6 mL/min flow rate. Fractions were
analyzed by LC-MS, and those with >90% homogeneity were collected,
lyophilized, and stored at −20 °C.

To extract the purified fusaricidin, the cells of P. polymyxa WLY78 and the fus mutants were grown in 100 mL of KL broth at 30 °C for 72 h with shaking at 200 rpm. The cells were collected by centrifugation at 12,000 rpm, 4 °C for 5 min, and extracted with 5 mL of methanol for 12 h. Then, the crude extracts were centrifuged at 12,000 rpm for 5 min to remove the cells and condensed by a vacuum freeze dryer, until ~2 mL of methanol remained. The crude extracts were fractioned by HPLC (Shimadzu LC-20AP) using the following method. A total of 1.5 mL of the methanol extract was injected into a C18 reversed-phase column (250 mm × 20 mm) with 90% acetonitrile (v/v) in 0.1% trifluoroacetic acid solution at a flow rate of 20 mL/min and detected by UV at 210 nm. The purified fractions were collected, air dried, and dissolved in 1 mL of methanol for antifungal activity assay. Then, 250 μL extracts of the purified fractions were dropped into the sterile iron rings (0.7 cm in diameter) on the PDA medium that had been mixed with 1 mL of the F. oxysporum f. sp. cucumerium spores (10⁶ CFU/mL). After cultivation at 28 °C for four days, the size of the inhibition zone was recorded.

Purification using RP-HPLC was performed using a Shimadzu High-Performance Liquid Chromatograph equipped with a SPD-M20A Prominence Photo Diode Array Detector and two LC-20AP pumps running lab solutions v5.97SP1. Preparative separations were performed on a Waters XBridge BEH300 Prep C18 column (5 μm, 19 × 150 mm) at a flow rate of 10 mL/min. The solvents used were water + 0.1% formic acid (solvent A) and HPLC-grade ACN + 0.1% formic acid (solvent B).