Combiflash rf 200i

The ionizable lipid C12-200 (1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol)) was synthesized by adding N¹-(2-(4-(2-aminoethyl)piperazin-1-yl)ethyl)ethane-1,2-diamine (1.00 g, 4.64 mmol, 1.0 equiv.), ethanol (10 mL), and 1,2-epoxydodecane (7.10 mL, 32.5 mmol, 7.0 equiv.) to a 50-mL round-bottom flask with a stir bar. The reaction was stirred at 80 °C for 48 h. Afterward, the ethanol was removed in vacuo and then diluted in 15 mL dichloromethane (DCM). The compound was purified using a CombiFlash (Teledyne ISCO, Lincoln, NE) using a 40 g RediSep Gold® silica gel flash column using a gradient mobile phase from 100% DCM to 20% DCM and 80% Ultra solution (75% DCM, 22% methanol, and 3% ammonium hydroxide) over 30 min. The solution was run purified in two runs, using half of the DCM mixture each time. C12-200 was isolated as a yellow-orange oil in 37% yield and characterized by ¹H NMR, ¹³C NMR, and LC-MS (SI Appendix, Figs. S12–S14).
¹H NMR and ¹³C NMR spectra were obtained on a NEO 400 MHz spectrometer using d-chloroform as the solvent. LC-MS was performed on an Agilent LCMS system equipped with UV–Vis and evaporative light scattering detectors (ELSD). Flash chromatography was performed on a Teledyne ISCO CombiFlash Rf-200i chromatography system equipped with UV-Vis and ELSD.

Combiflash R_f 200i (Teledyne ISCO) fitted with glass column and silica gel 60 RP-18 (40–63 μm) were used for purification of fractions. Shimadzu HPLC, LC 20AD with PDA detector fitted with either an analytical column (Discovery RP Amide C-16 Supelco 5 μm 250 × 4.6 mm) or semi-preparative (Discovery Amide C-16 Supelco 5 μm 250 × 10 mm) were used for analysis and purification of compounds. Column chromatography was carried out with Merck silica gel (100–200 mesh size). Thin layer chromatography (TLC) was done on TLC plates pre-coated with silica gel 60F254 (0.25 mm normal phase Merck). α-Glucosidase (Maltase EC 3.2.1.20) and p-nitro phenyl-α-d-glucopyranoside were purchased from Sisco Research Laboratory (SRL). Streptozotocin was purchased from Sisco Research Laboratories, Mumbai and metformin from Sigma Aldrich USA. 6-NBDG was procured from Invitrogen. NMR spectra were recorded on a Bruker Avance 500 MHz instrument with TMS as internal standard. Chemical shifts are expressed in δ values. Agilent 6520 Accurate mass Q-TOF/LC-MS was used to determine molecular weight. Absorbance was measured by Thermo Scientific Multiskan spectrometer. All other chemicals and reagents were of analytical grade.