pH and pKDeterminations by High-Resolution Solid-State¹³C NMR:  Acid−Base and Tautomeric Equilibria of Lyophilizedl-Histidine

Abstract

Acid-base properties of lyophilized powders of L-histidine have been systematically investigated using parent solutions at pH varying from 1.8 to 10. For the first time, high-resolution solid-state 13C NMR was shown to allow separate observation of all three acid-base pairs in the successive deprotonations of the carboxylic end, the imidazolium cation, and the terminal ammonio group of histidine. 1H CRAMPS NMR spectra directly visualize the absence of the N3-H(pi) tautomer in neutral and anionic species. Solid-state titration shifts are enlarged by approximately 1-4 ppm with respect to those measured in solution, permitting unambiguous observation of conjugate acid-base pairs. Calculated pK's from solid-state acid-to-base ratios r are found equal to those classically measured in solution at 0 degrees C with a similar ionic strength of 0.1 mol x dm(-3). This proves that natural-abundance 13C solid-state determinations of r can be used to measure pK's in parent solutions without recourse to full titration curves and subsequent curve-fitting procedures. Such an approach also leads to noninvasive characterizations of the acidity of lyophilized powders, i.e., to the prediction of in situ pH of products obtained after rehydration and solubilization of powders. These results show the possibility of measuring the pK of nonvolatile acidic substrates dissolved in any sublimable solvent through lyophilization of the investigated solutions; this leads the way to pH and pK determinations when electrochemical or spectrophotometric measurements are impossible or ambiguous, e.g., for concentrated solutions, polyacids, or mixtures of acidic solutes, and possibly to the establishment of pK scales in nonaqueous solvents and in melts.

On the magic-angle turning and phase-adjusted spinning sidebands experiments

“…The center-band-only spectrum at F 1 = 0 of the 2D MAT-PASS spectrum reveals the number of distinct carbon sites directly, while a sum of the slices allows reconstruction of the CP/MAS spectrum. The number of distinct resonances is higher than expected due to the presence of two polymorphs [40]. The other F 1 slices shows separated spinning sidebands with their intensities containing the CSA information.…”

Evaluation of the influence of amino acid composition on the propensity for collision-induced dissociation of model peptides using molecular dynamics simulations

“…There are two tautomeric forms of neutral forms of histidine to consider in which a proton is located on either the ␦ nitrogen (His-D) or nitrogen (His-E), and one protonated form of histidine (His ϩ 1) in which protons are on both the ␦ and nitrogens. The preferred neutral tautomer, His-D or His-E, is highly dependent on the local environment [37,38]. For each peptide represented in Figure 6 the proton donor is the N-terminal amine, except for histidine (His ϩ 1), lysine and arginine in which case the proton donor is the respective sidechain.…”

Effect of excipients on PLGA film degradation and the stability of an incorporated peptide

“…2.31 13 C Nuclear Magnetic Resonance ( 13 C NMR)-Both solution and solid-state NMR (ssNMR) have been used to estimate the pH and effective 'pH' of solid and semi-solid systems based upon the chemical shift changes of an ionizable functional group [17,18]. NMR is especially useful for establishing the protonation state and pKa of a functional group in either an organic solvent or in the solid-state [17,18].…”

“…NMR is especially useful for establishing the protonation state and pKa of a functional group in either an organic solvent or in the solid-state [17,18]. Here, 13 C NMR was employed because carbon NMR typically provides a chemical shift difference on the order of several ppm upon protonation [18], compared to shifts < 1 ppm for proton NMR [19].…”