NMR Analysis and Preparative HPLC Purification

Specific rotations were recorded using JASCO P-2000 digital polarimeter. Bruker DRX-400 NMR spectrometer with cryoprobe was used to record NMR spectra. The NMR spectrometer was equipped with 5 mm BBI (¹H, COSY, edited HSQC, and HMBC) or BBO (¹³C) probe head with z-gradients. The ¹H and ¹³C NMR chemical shifts were referenced to the residual solvent peaks for MeOH-d₄ at δ_H 3.31 and δ_C 49.0 ppm; or DMSO-d₆ at δ_H 2.50 and δ_C 39.5 ppm. Preparative RP-HPLC was performed using XTerra MS C₁₈ Prep column (19 × 300 mm, 10 µm) on an Agilent 1260 Infinity Preparative-Scale LCMS Purification System hyphenated with Agilent 6130B single quadrupole MS as a detector. LCMS data were recorded using Agilent UHPLC 1290 Infinity coupled to Agilent 6540 accurate-mass quadrupole time-of-flight (QTOF)-ESIMS. Standard gradient conditions of 98% H₂O (0.1% FA) to 100% CH₃CN (0.1% FA) were run over 8.6 min using an Acquity UPLC BEH C₁₈ (2.1 × 50 mm, 1.7 µm) column, all at a flow rate of 0.5 mL/min. The QTOF were set using the same parameter as previously reported [4 (link)].