Uflc system

NMR spectra were acquired on a 500 MHz Bruker Ascend Avance III HDTM equipped with a Prodigy ultra-high sensitivity multinuclear broadband CryoProbe operating at 500 and 125 MHz for 1H and 13C, respectively. They were referenced internally according to residual solvent signals. All ESI mass spectra were obtained from a Thermo Finnigan LCQ Deca XP (ThermoFisher Scientific, Waltham, MA). Highperformance liquid chromatography (HPLC) was performed on a Shimadzu UFLC system equipped with a 5 μm Phenomenex Luna C-18 column (Torrance, CA). Flash column chromatography was performed on 230-400 mesh silica gel supplied by Sigma-Aldrich (St. Louis, MO) with ACS grade solvent. R_f values are quoted for plates with thickness of 0.25 mm. The plates were visualized with iodine, UV, and phosphomolybdic acid reagents. All reactions were carried out under an argon atmosphere. All reagents were obtained commercially unless otherwise noted. Reactions were performed using glassware that was oven- dried at 120 °C. Air- and moisture sensitive liquids and solutions were transferred via syringe or stainless- steel cannula.

UV data were recorded on a Hewlett-Packard 8452A diode array spectrophotometer. Optical rotation measurements were made on an AUTOPOL III automatic polarimeter. IR spectra were recorded on a Shimadzu IRAffinity-1 FT-IR spectrometer. The LC-ESIMS analyses were performed on a Shimadzu UFLC system with a quadrupole mass spectrometer using a Phenomenex Kinetex C₁₈ column (3.0 mm × 75 mm, 2.6 μm) and MeCN−H₂O (with 0.1% HCOOH) gradient solvent system. HRESIMS spectra were measured using an Agilent 6538 Ultra High Definition (UHD) Accurate-Mass Q-TOF system. NMR spectra were obtained on Varian spectrometers (500 MHz for ¹H and 125 MHz for ¹³C at the University of Oklahoma; 600 MHz for ¹H and 150 MHz for ¹³C at the University of Florida) and Bruker Avance III spectrometer equipped with a nitrogen-cooled cryoprobe (600 MHz for ¹H and 150 MHz for ¹³C at Bruker Biospin Corp.) using DMSO-d₆ and CDCl₃ (Aldrich) as solvents. Column chromatography was conducted using Sephadex LH-20. HPLC was carried out using a Waters System equipped with a 1525 binary HPLC pump coupled to a 2998 PDA detector with a Phenomenex C₁₈ column (21.2 × 250 mm or 10 × 250 mm, 5 μm particle size). Glucose-¹³C₆,d₇, sodium acetate-d₃, and D₂O were purchased from Cambridge Isotope Laboratories, Inc. All other materials were purchased from Sigma-Aldrich.

HOHA-lactone (1, 1.4 mg, 10 μmol) in water (0.5 mL) was mixed with reduced L-glutathione (GSH, 6.14 mg, 20 μmol). The reaction mixture was stirred at room temperature for 2 h. The resulting mixture was then extracted three times with chloroform to remove unreacted HOHA-lactone. Then the remaining aqueous solution was separated by HPLC. Reverse-phase HPLC was conducted using a Shimadzu UFLC system equipped with a 5 μm 4.6 × 250 mm Phenomenex Luna C₁₈ column with a water-methanol supplemented with 0.1% formic acid gradient elution system at a flow rate of 1.0 mL/min, with effluent monitoring at 220 nm. Mobile phase A consisted of HPLC grade water containing 0.1% formic acid. Mobile phase B was HPLC grade methanol containing 0.1% formic acid. For purification of 4, 100 μL of reaction mixture was loaded onto the HPLC column for separation. HPLC gradient steps were as follows: 0-2.5 min, isocratic at 2% solvent B; 2.5-15 min, linear gradient from 2 to 100% solvent B; 15-20 min, isocratic at 100% solvent B; 20-21 min, linear gradient from 100 to 2% solvent B; 21-30 min, isocratic at 2% solvent B to give pure 4 (2 mg, 4.5 μmol, 45%). A representative HPLC chromatogram for purification of compound 4 is shown in Figure 2. The fraction between 11.00 to 13:00 min was collected.

UV data were recorded on a Hewlett Packard 8452A diode array spectrophotometer. Optical rotation measurements were made on an AUTOPOL^® III automatic polarimeter. The LC-ESIMS analyses were performed on a Shimadzu UFLC system with a quadrupole mass spectrometer using a Phenomenex Kinetex C₁₈ column (3.0 mm × 75 mm, 2.6 μm) and MeCN-H₂O (0.1% HCOOH) gradient solvent system. HRESIMS spectra were measured using an Agilent 6538 Ultra High Definition (UHD) Accurate-Mass Q-TOF system. NMR spectra were obtained on Varian spectrometers (500 MHz and 400 MHz for ¹H, and 100 MHz for ¹³C) using DMSO-d₆ and CDCl₃ as solvents. Column chromatography was conducted using silica gel and HP20SS. HPLC was performed on a Waters System equipped with a 1525 binary HPLC pump coupled to a 2998 PDA detector with a Phenomenex Gemini C₁₈ column (21.2 × 250 mm or 10 × 250 mm, 5 μm particle size).