1260 infinity instrument

The synthetic procedures and analysis were reproduced from the previously reported synthesis of JMC31 [21 (link)]. Electrospray ionization mass spectrometry (ESI-MS) was performed by an Agilent 1100 series liquid chromatograph system with an API-ES interface. Mass spectra were acquired in positive mode scanning over the mass range m/z of 150–1500. ¹H NMR and ¹³C NMR spectra were recorded on a Bruker Advance DPX400 operating at 400 MHz, using the residual signal of the deuterated solvent as the internal standard. The value of chemical shifts (δ) is given in ppm and coupling constants (J) are given in hertz (Hz). Splitting patterns are described as singlet (s), doublet (d), triplet (t), quartet (q), doublet of doublet (dd), and multiplet (m). The chemical purity of the final compound was determined using an Agilent 1260 Infinity instrument comprising a binary pump, an autosampler, a UV-DAD, and an ESI-MS detector. The chromatographic separation was performed with an Agilent Poroshell 120 EC-C18 column (4.6 mm × 100 mm, 2.7 μm), injecting 5 μL of the sample. Analyses were carried out with gradient elution, solvent A (0.1% formic acid in water), and solvent B (0.1% formic acid in acetonitrile), 90:10 to 10:90 over 6 min, at the flow rate of 2 mL/min, and a UV detector at 254 nm. The compound purity was ≥95.0%.

For HP-SEC, samples were analyzed with a TSKgel G3000SWxL column (7.8 × 300 mm, 5 μm). Chromatograms were recorded with a 1260 MWD VL UV detector, an Agilent 1260 Infinity instrument, and a 1260 ALS autosampler operating at a flow rate of 0.8 mL/min. The mobile phase consisted of 100 mM sodium phosphate dibasic buffer, 150 mM sodium chloride, and 0.05% (w/v) SDS at a pH of 7.0, and was filtered through a 0.2 μm filter prior to use.

Cell biomass was measured in terms of optical density at 600 nm (OD₆₀₀). The residual phenol and 4-chlorophenol contents were determined using high-performance liquid chromatography (HPLC) performed with an Agilent 1260 infinity instrument equipped with a reverse-phase poroshell 120 EC-C18 column (4.6 × 150 mm, 2.7 μm, Agilent). The mobile phase was a mixture of methanol and MillQ water (60:40 [vol/vol]) at a flow rate of 0.8 mL/min. The injection volumes for all samples were 5 µl and monitored at 210 nm with a variable-wavelength detector. LC-MS analysis was performed with a SHIMADZU LCMS-2020 instrument. Mass spectrometry was performed in ESI positive- and negative-ion modes with a scan range 50–500 m/z. The ESI-MS interface parameters were set as follows: drying gas (nitrogen) flow rate, 1.5 L/min; capillary column temperature, 350℃; spray voltage, 4.5 kV.

DSM6263 and CP-8 was initially cultured in M65 medium without NaCl for 12 h, Subsequently, 8% NaCl was introduced into the medium, and the cultures were further incubated for an additional 6 h. After centrifugation and bacterial pellet collection, rapid freezing was achieved using liquid nitrogen. The bacteria were then disrupted utilizing the liquid nitrogen grinding approach. A solution of 10% methanol was subsequently employed to resuspend the cell homogenate. cAMP was assayed using high-performance liquid chromatography (HPLC) performed with an Agilent 1260 infinity instrument equipped with a reverse-phase poroshell 120 EC-C18 column (4.6 × 150 mm, 2.7 μm, Agilent). The mobile phase was a mixture of methanol and 100mM KH₂PO₄ (10:90 [vol/vol]) at a flow rate of 1 mL/min. The injection volumes for all samples were 5 µl, respectively and monitored at 254 nm with a variable-wavelength detector.