Underlining the Importance of Peripheral Protic Functional Groups to Enhance the Proton Exchange of Gd-Based MRI Contrast Agents

In this study, we report the synthesis and the equilibrium, kinetic, relaxation, and structural properties of two new GdIII complexes based on modified 10-(2-hydroxypropyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (HPDO3A) designed to modulate the relaxivity at acidic and basic pH due to intra- and intermolecular proton exchange. The presence of a carboxylic or ester moieties in place of the methyl group of HPDO3A allowed differentiation of a protic and nonprotic functional group, highlighting the importance of the formation of an intramolecular hydrogen bond between the coordinated hydroxyl and the carboxylate groups for proton exchange (kH = 1.5 × 1011 M–1 s–1, kOH = 1.7 × 109 M–1 s–1). The determination of the thermodynamic stability and kinetic inertness of the GdIII complexes confirmed that the modification of peripheral groups does not significantly affect the coordination environment and thus the stability (log KGdL = 19.26, t1/2 = 2.14 × 107 hours, pH = 7.4, 0.15 M NaCl, 25 °C). The relaxivity (r1) was measured as a function of pH to investigate the proton exchange kinetics, and as a function of the magnetic field strength to extrapolate the relaxometric parameters (r1GdL1 = 4.7 mM–1 s–1 and r1GdL2 = 5.1 mM–1 s–1 at 20 MHz, 25 °C, and pH 7.4). Finally, the X-ray crystal structure of the complex crystallized at basic pH showed the formation of a tetranuclear dimer with alkoxide and hydroxide groups bridging the GdIII ions.

Standard deviations (3) are shown in parentheses. By taking into account the protonation sequence of the macrocyclic DOTA-like ligands determined by spectroscopy and potentiometry methods, 1-3 it is assumed that the first and second protonation of L2 occur at two opposite ring nitrogen atoms, whereas the third protonation process takes place at one of the carboxylate groups attached to the non-protonated ring nitrogen atoms. Fourth proton is localized on the carboxylate group of the 2hydroxypropanoic side chain, whereas further protonation of L2 occurs at the non-protonated carboxylate pendant arms. Comparison of the protonation constants (Table S1)  Scheme S1. Macrocyclic ligands discussed in the present work. The stability and protonation constants of Ca II -, Zn II -, Cu II -and Gd III -complexes of L2, expressed by Eqs. (S2) and (S3), were determined by pH-potentiometry and spectrophotometry at 25 ºC in 0.15 M NaCl solution.
where i=0, 1, 2, 3. The KML and KMHiL values of Ca II -, Zn II -and Cu II -L2 complexes were calculated from the pH-potentiometric titration data obtained at 1:1 metal to ligand concentration ratios. In the calculations, the best fitting of the mL NaOH -pH data was obtained by assuming the formation of ML, MHL, MH2L and MH3L species. The stability constant of GdL2 was calculated from the equilibrium data obtained by the "out-of-cell" technique due to the slow complexation reaction.
The formation of lanthanide(III)-complexes with DOTA and DOTA derivatives takes place via the diprotonated "out-of-cage" intermediate species (e.g. *Ln(H2DOTA)), in which the Ln III -ion is coordinated by the carboxylate groups, whereas two opposite ring nitrogens are protonated. [6][7][8][9][10] Tipically, the formation of the "in cage" Ln(DOTA)and LnDOTA-like complexes occurs by the slow deprotonation of the ring nitrogens, which is followed by the penetration of the Ln III -ion into the N and O donors coordination cage of the ligand. In order to calculate reliable equilibrium constants, the presence of the Ln(H2DOTA) + intermediate, the free Ln 3+ ion and the final Ln(DOTA)complex should be considered in the equilibrium Ln III -DOTA system. 11,12 The formation and For the complete characterization of the Gd 3+ -L2 system, the GdL2 complex (which is formed completely at about pH=3.5) was titrated with 0.2 M NaOH solution in the pH range 3.5 -12.0.
During these titrations, base consumption was observed at about pH>3.5 and pH>8.0, which indicated the deprotonation of the Gd(HL) and GdL species, respectively. These processes correspond the dissociation of H + ion from the carboxylate and the alcoholic -OH group of the 2-hydroxypropanoic pendant arm (Eqs. (S3) and (S4)). A pH-potentiometric titration of GdL1 complex with 0.2 M NaOH solution was also performed to determine the deprotonation constant of the hydroxyl group in the methyl 2-hydroxypropanoate pendant arm (logKGd(L)H-1, Table S1).
By taking into account the protonation constants of the ligand L2, the stability constant of the *Gd(H2L2) intermediate and the stability and protonation constant of the GdL2 complex, the species distribution of the Gd 3+ -L2 system was calculated ( Figure S1). The stability and protonation constants of the Ca II -, Zn II -, Cu II -and Gd III -L2 complexes are shown and compared with those of HPADO3A, HPDO3A, DOTA and BT-DO3A complexes in Table S1.
The stability constants of the Ca II , Zn II , Cu II and Gd III complexes formed with L2, HPADO3A and BT-DO3A (Table S1) (6)

Kinetic inertness of GdL2
In order to investigate the kinetic inertness of GdL2, the dissociation reactions of GdL2 were followed by 1 H-NMR relaxometry in the presence of large acid excess ([HCl]= 0.01 -1.0 M) to guarantee the pseudo-first-order kinetic conditions. The R1 obs values as a function of time for the dissociation reactions of GdL1 are shown in Figure S3.

[H + ] (mol/dm 3 )
In the presence of HCl excess, the dissociation of GdL2 can be treated as a pseudo-first-order process and the rate of the reaction can be expressed by Eq. (S7)