JP-2022534576-A5 -

Dates

Publication Date

20230525

Application Date

20200525

Description

(1.14. Intermediate 14) To a solution of intermediate 13 (10 g, 39.1 mmol, 1 equivalent) stirred in 240 mL of THF at 0°C, LiHMDS (1 N in THF, 59 mL, 58.6 mmol, 1.5 equivalents) is added. The resulting mixture is stirred at 0°C for 45 minutes, and 2,2,2-trifluoroethyltrifluoromethanesulfonate (CAS# 6226-25-1; 8.44 mL, 58.6 mmol, 1.5 equivalents) is added dropwise at 0°C. The reaction mixture is slowly heated to RT and stirred at RT for 22 hours. The reaction mixture is quenched with water and brine. The THF is evaporated, and the aqueous layer is extracted with ELISA. The combined organic layers are dried over anhydrous Na₂SO₄ , filtered, and concentrated under vacuum. The residue is purified by flash chromatography on silica gel (eluting with 0–1% MeOH in DCM) to obtain intermediate 14. (2.2.1.2. 4 IL-10 ELISA) A 96-well Immulon 2HB plate (Thermo Electron, catalog number 3455) is coated with 40 μL of capture antibody (final concentration 2 μg/mL, diluted in Tris buffer (50 mM Tris; 150 mM NaCl; pH 9 (adjusted with HCl)) and stored overnight at 4°C. The next day, the plate is washed three times with PBST, followed by the addition of 200 μL of blocking buffer (1% BSA + 5% sucrose in PBS-T). After incubation at 37°C for 30 minutes, the plate is washed three times with PBST, and 100 μL of standard material or sample is added (IL-10 standard curve samples are prepared using 1/2 serial dilutions starting from 1000 pg/mL; dilutions are prepared using dilution buffer: PBS + 1%). (Prepared in BSA). After incubation at 37°C for 1 hour, wash the plate three times with PBST, then add 100 μL of detection antibody (BD Pharmingen, catalog no. 554499) (final concentration 0.25 μg/mL, diluted in Tris buffer), and incubate the plate at room temperature for at least 2 hours. Wash the plate three times with PBST, then add 100 μL of Strep-HRP conjugate (final concentration 0.5 μg/mL, diluted in dilution buffer). Incubate the plate in the dark at 37°C for 30 minutes. Wash the plate three times with PBST. Prepare the substrate solution: for a total volume of 20 mL, 18 mL of H₂O ; 2 mL of citrate acetate buffer; 200 μL of TMB mix (tetramethyl benzidine (TMB) 101 mg, DMSO 10 mL, stored at 4°C); 2.5 μL of 30% H₂O Mix in O2 . Add 100 μL of substrate solution to each well and incubate until a vivid blue color is produced. Stop the reaction by adding 50 μL of 1 M H2SO4 , and then measure the absorbance at 450 nm using SpectraMax i3 Molecular Devices.