Lc 6ad pump

Mucin disks were prepared using the method of Tsuchiya et al. [25 ]. Four-hundred microliters of 10% mucin solution was spread on a 25-mm diameter filter paper. The filter paper was dried at room temperature for 24 h. An artificial saliva solution (pH 6.8) consisting of 0.8% NaCl, 0.019% KH₂PO₄, and 0.238% Na₂HPO₄ was prepared. The mucin disk was moistened with artificial saliva, and 3 mg of IM bulk powder or the IM microparticles were mounted on the mucin disk, and the mucin disk was dried at room temperature for 5 min. Each mucin disk with sample was fixed to a slide glass with a clip and immersed in 150 mL of artificial saliva at 37 °C and incubated with shaking at 50 rpm. Mucin disks were taken out at 5, 10, and 15 min and the amount of IM remaining on the disk was measured using high-performance liquid chromatography (HPLC). The HPLC system consisted of an LC-6AD pump (Shimadzu Corporation, Kyoto, Japan) and a Chromato-PRO (Run Time Corporation, Tokyo, Japan) equipped with a Capcell Pak C18 MG II column (4.6 × 250 mm, OSAKA SODA CO., LTD., Osaka, Japan) and an SPD-20AV UV detector (Shimadzu Corporation). Chromatography was carried out at 40 °C. The mobile phase comprised 60% (v/v) acetonitrile in 0.02 M sodium acetate buffer adjusted to pH 3.6 using orthophosphoric acid. The flow rate was 1 mL/min. The detection wavelength was 320 nm.

The reaction progress was monitored using thin-layer chromatography (TLC) on silica gel 60 F₂₅₄ (0.25 mm, Merck, Darmstadt, Germany). Column chromatography was performed using silica gel 60 (0.04–0.063 mm, Merck). Melting points were determined using a Yanaco (Tokyo, Japan) micro-melting-point apparatus without correction. HPLC was performed using an EYELA Preparative LC system [VSP-3050 pump, UV-9000 spectrometric detector, LiChrosorb RP-18 column (10 μm, 25 mm × 300 mm)] (Tokyo Rikakikai Co. Ltd., Tokyo, Japan) and a Shimadzu LC system [LC-6 AD pump, SPD-20A UV spectrometric detector, Mightysil RP-18 column (5 μm, 20 mm × 250 mm)] (Kyoto, Japan). The NMR spectra were recorded with a JEOL JNM-LA400 spectrometer (Tokyo, Japan). The chemical shifts were expressed in ppm, downfield from TMS. The mass spectra were collected using a JEOL JMS-SX102A mass spectrometer (Tokyo, Japan).

petroleum ether, CHCl₃, EtOAc, and n-BuOH were used to extract the culture filtrate of HN09 sequentially three times to generate dried petroleum ether-soluble (29.87 g), CHCl₃-soluble (16.03 g), EtOAc-soluble (30.6 g), and n-butyl alcohol-soluble (80.15 g) extracts. The EtOAc-soluble extract was subjected to passage over a silica gel column (100–200 mesh) which was eluted with CHCl₃-methanol mixtures of increasing polarities (100:0–90:10–80:20–70:30–60:40–50:50, v/v). A total of 10 fractions (E1-E10) were obtained. Fraction E4 (7.4 g), which was obtained by elution with CHCl₃–MeOH (80:20), was rechromatographed on a Develosil ODS (10 μm, Nomura Chemical Co. Ltd., Japan) column. Moreover, 16 subfractions (E4–1–E4–16) was obtained by eluting Fraction E4 with MeOH–H₂O mixtures (3:7, 1:1, and 7:3). preparative HPLC was used to separate the subfraction E4–1, which was run with a Shimadzu RID-10A refractive index detector and a Shimadzu LC-6 AD pump by taking a YMC-pack ODS-A C18 column (5 μm, 250 × 20 mm) and MeOH-water mixture (7:93, v/v) as the mobile phase to generate B16 (2.86 g) and B17 (1.76 g).

Scale-up reactions have a total volume of 60 mL, containing 100 μg/mL purified UGT109A1, 25 mM Tris-HCl (pH 8.0), 10 mM substrate dissolved in DMSO, and 50 mM UDPG. The reactions were performed at 37 °C for 12 h and terminated by adding 60 mL methanol. Then, the reactants were evaporated under reduced pressure distillation. The remaining residues were resuspended in 5.0-10.0 mL methanol, filtered by a 0.22 μm filter, and then purified by semi-preparative HPLC. Semi-preparative HPLC was performed on an HPLC system equipped with a Shimadzu LC-6AD pump and a Shimadzu SPD-20A prominence UV-VIS detector (Shimadzu Corporation, Kyoto, Japan) using a C₁₈ column (10 mm × 250 mm, 5 μm particles, CAPCELL PAK, Japan). The HPLC conditions are described in detail in Supplementary Information Table S7.