Fig. 7
Determination of the pKa value of M23 by NMR. (A) Resolved, high-field portion of the 700 MHz 1H NMR spectrum of 300 µM M23 (chemical structure shown on top) measured in the pH range of 2 to 13 in 50 mM sodium phosphate, 100 mM sodium chloride (80% (v/v) H2O, 10% (v/v) D2O, 10% (v/v) DMSO-d6). Sodium 2,2-dimethyl-2-silapentane-5-sulfonate (DSS) was used for chemical shift referencing and calibrated to a zero ppm value. (B) Chemical shift values of reporter protons (labeled as 1’’’ and 2’’, marked by arrows in the chemical structure) were plotted against pH. The pKa value was calculated by fitting to the Henderson-Hasselbalch equation. The pKa value of the secondary amine is 6.69 ± 0.01 and 6.63 ± 0.02 as indicated by protons 1’’’ and 2’’, respectively.
