Method for determining pKa of invisible small molecules using spatially resolved 1H NMR through pH gradient and proton absorption parameters alone Haider Hussain, Matthew Wallace and Yaroslav Khimyak University of East Anglia, UK The acid dissociation constant ( K a or its negative logarithm p K a ) of a molecule is an essential property that has a significant impact on itschemical and biological activity and is used in drug discovery for predicting the drug-target interactions of pharmaceutical molecules and their respective solubility. This can be seen in characterising active pharmaceutical ingredients (API) where p K a is used to estimate the extent of API ionisation which helps predict the absorption, distribution, metabolism, and excretion of API in biological systems and the environment. 1 NMR spectroscopy is a powerful tool for determining the p K a of molecules in a non-invasive manner using chemical shift information and the fact that the average chemical shifts of all the measurable NMR-active nuclei are expected to reflect the fractional protonation of each ionisable group of a molecule. 2 Typically NMR p K a measurements are done using multiple 1D NMR experiments which have varying concentrations of the base and then measuring the chemical shift of the analyte as a function of pH Which can be time consuming and laborious. However, we managed to obtain p K a values using pH gradated NMR samples and 1 H chemical shift imaging (CSI) to obtain titration curves in a “single shot” NMR experiment. 3 This greatly cuts down the time and material needed to do the experiment however it is still limited to studying samples with observed analyte chemical shift which is difficult to obtain in inorganic or polymeric systems. Here, we present a method for obtainingp K a of NMR invisible molecules using theCSI 2D 1 H NMR technique using pH gradated NMR tube and the number of protons absorbed by a base alone without the need for observing the analyte’s chemical shift. Wealso implement a new fitting function for the NMR data that allows for obtaining of p K a and concentration of the analyte through calculating the slope and y-intercept of a linear plot. We investigated a collection of small molecules,both visible and invisible, of a wide range of p K a values using a variety of base indicators and obtained p K a values within 0.4 units of literature value. This technique has potential for application on obtaining p K a of polymeric and colloidal systems despite their poor NMR visibility.
Scheme 1. (a) Proton absorption (PA) equations from an acid and a base point of view and method of determining pK a analytes using 2D 1 H CSI. (b) equation as a function of H + with graphs for 10mM Boric acid with 2-methly imidazole (2mi) as base indicator, with the fitted pK a and wider literature pK a with good agreement References 1. Sandra, H. Alka, M. Dragana and K. Marija, TrAC Trends in Analytical Chemistry. , 2007, 96 , 1043-1061. 2. Gift, S. Stewart and P. Kwete, Journal of Chemical Education, 2012, 89 , 1458-1460 3. Wallace, D. J. Adams and J. A. Iggo, Anal. Chem. , 2018, 90 , 4160-4166.
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