NMR measurement of pKa of water-soluble ionizable lipid analogs

The pH dependence of proton NMR chemical shifts was used to measure the pKa’s of the ionizable lipid water-soluble analogs following published methods^27,55. Chemical shifts of piperazine and imidazole were used as internal pH indicators. Solutions were prepared with 100 mM KCl, 2 mM piperazine, 2 mM imidazole, and 5 mM water-soluble ionizable lipid analog in 95% H₂O-5% D₂O. This solution was split into two equal volumes and one titrated to a lower pH (e.g. 6) using 0.1 M HCl and the other to an upper pH (e.g. 12) using 0.1 M NaOH. Intermediate pH values were obtained by mixing different proportions of these two solutions. NMR measurements were performed on a Bruker 400 MHz spectrometer where ¹H spectra were acquired at each of ~20 pH values ranging from the lower to upper pH (e.g. 6–12). Chemical shifts from piperazine and imidazole were then used to calculate the pH of each solution according to published methods^27,55 and the chemical shifts of the N-terminal amine group protons of the water-soluble ionizable lipid analogs were fit to the Henderson–Hasselbalch equation δ=δmax−δmax−δmin/1+10pKa−pH to provide the pKa of the N,N-Dimethylamine moiety in the different head groups (Spectra in Supplementary Fig. 1).