NMR Measurement of pKa of Nitrogen Groups

Abstract

Messenger RNA has recently attracted great interest due to in vivo evidence of high efficacy in vaccine and cancer applications. A delivery system is required for the mRNA to protect it from enzymatic attack and facilitate cellular entry and intracellular release and translation of the payload. Currently, solid Lipid NanoParticles (LNPs) are the most effective delivery system for mRNA. Studies to date have revealed that the ionization behavior of the nitrogen-containing cationic lipid in LNPs is key to intracellular release. Direct measurement of pKa of the cationic lipids has not been reported to date but would be helpful in the rational design of novel systems. Nuclear magnetic resonance spectroscopy (NMR) has proven to be advantageous in determining the pKa of these lipids because it can measure mixtures with impurities, as mole fractions are used instead of acid concentration. To develop an accurate method of measuring pKa with NMR, sample solutions of pyridine were prepared with a small amount of D2O as an NMR standard. This sample solution was titrated to a range of pH levels centered around the theoretical pKa of pyridine. 0.7 mL of each solution at a defined pH was added to an NMR tube, and 1H with water suppression NMR spectra were acquired. The chemical shift of individual peaks in the NMR spectra were plotted as a function of pH and fit a sinusoidal line. A pKa of 5.373 was calculated, which is similar to the theoretical value, thus confirming that NMR is an accurate technique to determine the pKa of these systems.