Protonation of carboxyl groups in EuDOTA-tetraamide complexes results in catalytic prototropic exchange and quenching of the CEST signal

The CEST properties of EuDOTA-tetraamide complexes bearing pendant carboxylate and carboxyl ethyl esters were measured as a function of pH. The CEST signal from the Eu³⁺-bound water molecule decreased in intensity between pH 8.5 and 4.5 while the proton exchange rates (<i>k</i>_ex) increased over this same pH range. In comparison, the CEST signal in the corresponding carboxyl ester derivatives was nearly constant. Both observations are consistent with stepwise protonation of the four carboxylic acid groups over this same pH range. This indicates that negative charges on the carboxyl groups above pH 6 facilitate the formation of a strong hydrogen-bonding network in the coordination second sphere above the single Eu³⁺-bound water molecule, thereby decreasing prototropic exchange of protons on the bound water molecule with bulk water protons. The percentage of square antiprismatic versus twisted square antiprism coordination isomers also decreased as the appended carboxylic acid groups were positioned further away from the amide. The net effect of lowering the pH was an overall increase in <i>k</i>_ex and a quenching of the CEST signal

The effective magnetic moment (μ_eff) [9] of the four lanthanide ion complexes and the measured T₁ (sec) ± s.d. of bulk water in 20 mM samples of LnDOTA-(gly)₄⁻.

<p>All data were collected at 9.4 T, 310 K on samples adjusted to pH 7.5.</p

CEST spectra of TmDOTA-(gly)₄⁻ collected at different pH values.

<p>Each spectrum was recorded at 9.4 T on 20 mM samples at pH 7.5 and 310 K. (B₁ = 21.2 µT, presaturation time = 2 s).</p

Amide proton CEST intensity <i>versus</i> pH for the four different LnDOTA-(gly)₄⁻ complexes.

<p>Each amide proton CEST intensity was recorded on 20 mM samples at 9.4 T, 310 K and the indicated pH after application of a frequency-selection presaturation pulse set to the frequency of the exchanging –NH proton characteristic of each complex (B₁ = 21.2 µT, presaturation time = 4 s (YbDOTA-(gly)₄⁻) or 2 s (TmDOTA-(gly)₄⁻, TbDOTA-(gly)₄⁻, DyDOTA-(gly)₄⁻).</p

Hydrodynamic diameter, polydispersity index and thulium-phosphorus ratio of the liposomes at different concentrations of the encapsulated TmDOTA-(gly)₄⁻.

<p>Hydrodynamic diameter, polydispersity index and thulium-phosphorus ratio of the liposomes at different concentrations of the encapsulated TmDOTA-(gly)₄⁻.</p

Amide proton CEST intensity <i>versus</i> pH for three different concentrations of TmDOTA-(gly)₄⁻.

<p>No lipid was present in this experiment. The amide proton CEST intensity was measured at 9.4 T and 310 K using a B₁ = 7.0 µT and a presaturation time of 6 s.</p

CEST spectra of the four LnDOTA-(gly)₄ complexes.

<p>Each complex (20 mM, pH 7.5, 310 K) shows a different amide proton chemical shift with respect to the bulk water (δH₂O = 0 ppm). The spectra were collected at 9.4 T using a frequency-selective, presaturation pulse over the frequency range ±100 ppm (B₁ = 21.2 µT, 2 s for Tm-, Dy-, Tb-DOTA-(gly)₄⁻ and 4 s for YbDOTA-(gly)₄⁻).</p

<i>In vitro</i> stability of a liposomal suspension at different pH values (n = 2).

<p>TmDOTA-(gly)₄⁻ was measured analytically in the extraliposomal medium after incubation of liposomes at 298 K for 24 hr at several different pH values.</p

Plot of amount of TmDOTA-(gly)₄⁻ released from liposomes (%) as a function of time.

<p>TmDOTA-(gly)₄⁻ was measured analytically in the extraliposomal medium during incubation of liposomes at 275 K, 298 K and 310 K over a span of 48 hr.</p

Comparison of amide proton CEST intensities <i>versus</i> pH for free and liposome-encapsulated TmDOTA-(gly)₄⁻.

<p>The amide proton CEST intensity for free TmDOTA-(gly)₄⁻ sample (3 mM, no lipid, same data as that shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027370#pone-0027370-g008" target="_blank">Fig. 8</a>) and liposomal-encapsulated TmDOTA-(gly)₄⁻ (intraliposomal concentrations of 75 mM and 150 mM) were measured as a function of pH at 9.4 T and 310 K using a B₁ = 7.0 µT and a presaturation time of 6 s. The number of liposomes in the later two samples was adjusted so that the total concentration of TmDOTA-(gly)₄⁻ in each sample was 3 mM when averaged over the entire volume.</p