Exploiting the Proton Exchange as an Additional Route to Enhance

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Cite This: Inorg. Chem. XXXX, XXX, XXX−XXX

Exploiting the Proton Exchange as an Additional Route to Enhance the Relaxivity of Paramagnetic MRI Contrast Agents Silvio Aime,† Simona Baroni,† Daniela Delli Castelli,† Ernő Brücher,‡ István Fábián,‡,§ Sonia Colombo Serra,∥ Alberto Fringuello Mingo,∥ Roberta Napolitano,∥ Luciano Lattuada,∥ Fabio Tedoldi,∥ and Zsolt Baranyai*,∥ †

Department of Molecular Biotechnologies and Health Sciences, Molecular Imaging Center, University of Torino, Via Nizza 52, 10126 Torino, Italy ‡ Department of Inorganic and Analytical Chemistry, Faculty of Science and Technology, §MTA-DE Redox and Homogeneous Catalytic Reaction Mechanisms Research Group, University of Debrecen, Debrecen, Egyetem tér 1, H-4032, Hungary ∥ Bracco Imaging Spa, Bracco Research Centre, Via Ribes 5, 10010 Colleretto Giacosa (TO), Italy ABSTRACT: The relaxivity of Gd(HP-DO3A) was studied as a function of pH and buffer composition in order to identify the main factors of the observed relaxation enhancement due to the exchange of the coordinated hydroxyl proton. It was established that the paramagnetic relaxation time, T1M, of the coordinated hydroxyl proton is about 50% shorter than that of the protons in the coordinated water molecule. The control of the pK of the coordinated alcoholic −OH moiety in the ligand is fundamental to utilize the proton exchange enhanced relaxivity under physio/pathologic conditions. A new derivative of Gd(HP-DO3A) was synthesized by replacing the −CH3 group with a −CF3 moiety. In this complex, the −OH group becomes more acidic. Consequently, the maximum contribution of the proton exchange to the relaxivity is shifted to a lower pH region with the fluorinated ligand.

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administration of GBCA.14−16 A possible route to deal with this issue is the reduction of the administered doses. This goal has generated renewed interest in improving the attainable relaxivity of GBCAs.17 Among others, proton exchange reactions between functionalities coordinated to the metal center and the bulk water molecules may contribute to enhance the relaxivity. Thus, a thorough mechanistic interpretation of such processes is of utmost importance. Actually, Gd(HPDO3A) (Scheme 1) contains a coordinated − OH moiety that acts as a source of proton exchange enhanced relaxivity, however, at pH values higher than the patho-physiological ones.18,19 Herein we show that an in-depth understanding of

INTRODUCTION Gadolinium(III) complexes have earlier been identified as candidates of choice for acting as contrast agents (CAs) in Magnetic Resonance Imaging (MRI).1−3 Gadolinium-based CA (GBCA) can induce large changes in the water proton relaxation times (T1 and T2) due to the large paramagnetism (7 unpaired electrons) and the long electronic relaxation time of the Gd3+ ion.4 The theory of paramagnetic relaxation was well established in the seventies of the previous century and the structural and dynamic factors influencing the relaxivity (i.e., the relaxation enhancement induced on solvent water protons by a given GBCA at 1 mM concentration) were clearly identified.1,4,5 Along the last three decades, chemists have addressed the task of improving the relaxation enhancement capability of GBCAs by designing new coordination architectures, multimeric derivatives and supramolecular adducts.1,3,6,7 These studies have shown how the elongation of the molecular reorientational time (either through the formation of supramolecular systems), in the presence of an optimized exchange lifetime of the coordinated water molecule, may yield significant enhancements of the attainable relaxivity. Other studies have addressed the design of complexes containing more than one coordinated water molecules (q = 2−3) as the increase of the number of water molecules in the innercoordination sphere improves the relaxivity.8−13 Recently, concern was raised due to the tiny amounts of GdIII retained in the tissues of patients undergone to the © XXXX American Chemical Society

Scheme 1. Structures of the Ligands H3HP-DO3A and H3CF3-HP-DO3Aa

a

H3HP-DO3A = 10-(2-hydroxypropyl)-1,4,7,-10-tetraazacyclododecane-1,4,7-triacetic acid; H3CF3-HP-DO3A = 10-[3,3,3-trifluoro-2hydroxypropyl]-1,4,7,10-tetraazacyclododecan-1,4,7-triacetic acid.

Received: February 28, 2018

A

DOI: 10.1021/acs.inorgchem.8b00521 Inorg. Chem. XXXX, XXX, XXX−XXX

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Inorganic Chemistry

computer program Micromath Scientist, version 2.0 (Salt Lake City, UT, U.S.A.).

the intimate details of the proton exchange occurring in Gd(HP-DO3A) provides relevant insights to the design of novel systems which are able to boost the relaxivity in the pH range of interest for medical applications.

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RESULTS AND DISCUSSION In principle, GdIII complexes enhance the nuclear relaxation rate of solvent water protons through the modulation of the dipolar interaction between the unpaired electrons of the metal ion and the nuclear spins of protons via three processes (Figure 1): (i) the diffusion of water molecules in the proximity of the

EXPERIMENTAL SECTION

Chemicals. The chemicals were of the highest analytical grade. GdCl3 solution were prepared by dissolving Gd2O3 (99.9%, Fluka) in 6 M HCl and by evaporating the excess acid. The concentrations of the GdCl3 stock solutions were determined by complexometric titration with standardized Na2H2EDTA solution, using xylenol-orange indicator. H3HP-DO3A ligand was provided by Bracco Imaging S.p.a.. H3CF3-HP-DO3A has been prepared as described earlier.20,21 Gd(HP-DO3A) and Gd(CF3-HP-DO3A) solutions were prepared by mixing equivalent amounts of GdCl3 and H3HP-DO3A or H3CF3-HPDO3A solutions. The pH of the solutions of the complexes was set to about 6.0 by addition of calculated amounts of 0.2 M NaOH solution. The concentration of the H3HP-DO3A and H3CF3-HP-DO3A ligands was determined by pH-potentiometric titration in the presence and in the absence of a large (40-fold) excess of CaCl2. The pHpotentiometric titrations were carried out using standardized 0.2 M NaOH. Equilibrium Measurements. The protonation constant of ligands and complexes Gd(HP-DO3A) and Gd(CF3-HP-DO3A), as well as CO32−, PO43−, and HEPES buffers were determined by pHpotentiometric titration, as described earlier.22 The concentrations of the GdIII complexes and buffers were around 0.002 M. For pH measurements and titrations, Metrohm 932 pH meter, Metrohm 665 Dosimat autoburet, and Metrohm-6.0234.110 combined electrode were used. The titrated solutions (6 mL) were thermostated at 298 K. The samples were stirred with a magnetic stirrer and to avoid the effect of ambient CO2, N2 gas was bubbled through the solutions. The ionic strength of the solutions was kept constant with 0.15 M NaCl. The titrations were carried out in the pH range 1.7−12.0. For the calibration of the pH meter, 0.05 M KH-phthalate and 0.01 M borax buffers were used.23 For the calculation of the H+ concentration from the measured pH values, the method proposed by Irving et al. was used.24 A 0.01 M HCl solution was titrated with the standardized NaOH solution at 0.15 M NaCl ionic strength. The differences (A) between the measured (pHread) and calculated pH (−log[H+]) values were used to obtain the equilibrium H+ concentration from the pHread values in the titration experiments (A = 0.036). In general, the pH corresponds to−log[H+] through all this paper. For the equilibrium calculations, the stoichiometric water ionic product (pKw) was also needed to calculate [OH−] values under basic conditions. The VNaOH − pHread data pairs of the HCl − NaOH titration obtained in the pH range 10.5−12.0 were used to calculate the pKw value (pKw = 13.85). The equilibrium constants were calculated by the use of the program PSEQUAD.25 1 H NMR Relaxometry. The relaxivity values were calculated from the longitudinal relaxation time of water protons (T1) measured with a Bruker Avance III 400 (9.4 T) NMR spectrometer equipped with BB inverse z gradient probe (5 mm). The temperature of the sample holder was kept constant (298 K) with a thermostated air stream. The longitudinal relaxation times were measured with the “inversion recovery” method (180° − τ − 90°) by using eight different τ values. The measurements were carried out in a 1 mM nondeuterated aqueous solutions of the Gd(HP-DO3A) and Gd(CF3-HP-DO3A) complexes, respectively. The relaxivity values were given as r1 = 1/T1p + 1/T1w, where T1p and T1w are the relaxation times of the bulk water protons in the presence and absence of GdIII complex. The pHdependent relaxivity measurements of Gd(HP-DO3A) and Gd(CF3HP-DO3A) complexes were carried out by direct titration of the samples (5.5 < pH < 12.5) in the presence and absence of HCO3−/ CO32−, H2PO4−/HPO42−/PO43− and HEPES buffers; [GdL] = 1.0 mM, 0.15 M NaCl and 25 °C. The relaxivity values of Gd(CF3-HPDO3A) complex were also measured at pH = 7.4 and 8.0 in Seronorm (SERO). The pH was adjusted by stepwise addition of concentrated NaOH or HCl solution. Calculations were performed with the

Figure 1. Contributions to the relaxivity of the Gd(HP-DO3A) complex.

paramagnetic complex (outer-sphere term: r1os); (ii) the exchange of water from the coordination site(s) to the “bulk” (inner-sphere term: r1is); (iii) the proton exchange involving the mobile protons in the complex (r1pr).3,4,26 The relaxivity of Gd(HP-DO3A) is determined by the r1is, os r1 , and r1pr terms (eq 1) r1p = r1is + r1os + r1pr

(1)

In the case of Gd(HP-DO3A), the last term of eq 1 corresponds to the −OH proton exchange and can be expressed as follows.27−29 c 1 r1pr = H 111.1 T1Pr + τpr (2) where c is the concentration of the complex, TH1Pr and τpr are the longitudinal relaxation time and the lifetime of the −OH proton, respectively. The relaxation enhancement brought by Gd(HP-DO3A) exhibits distinct pH dependence (Figure 2).

Figure 2. Relaxivity of Gd(HP-DO3A) as a function of pH. Symbols and solid lines represent experimental and calculated relaxivity values, respectively. Calculations have been performed by the use of eq 5 (400 MHz, 0.15 M NaCl, 298 K).

In the pH range 6.0−7.5, the relaxivity is constant. At pH > 7.5, the relaxivity increases as a function of pH to reach a maximum at pH 10.5. Since the random translation diffusion of the water molecules in the surroundings of the paramagnetic center and the exchange of the water molecule in the first coordination sphere of the paramagnetic center with the bulk are pH-independent, both r1is and r1os terms are pHB

DOI: 10.1021/acs.inorgchem.8b00521 Inorg. Chem. XXXX, XXX, XXX−XXX

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Table 1. Kinetic and Relaxation Parameters for the Proton Exchange Reactions of Gd(HP-DO3A) (400 MHz, 0.15 M NaCl, 298 K) GdL is+os r1 (mM−1 s−1) Gd(L)H‑1 is+os r1 (mM−1 s−1) TH1Pr × 106 (s) k1 (M−1 s−1)

3.93 ± 0.02 4.07 ± 0.02 4.3 ± 0.1 (5.2 ± 0.4) × 109 (OH−)

4.9 ± 0.1 (9 ± 1) × 107 (CO32−)

Gd(L)H−1 + H+ ⇌ GdL [GdL] [Gd(L)H−1][H+]

(3)

The proton exchange process between the −OH proton and the bulk water, is described by the following equation: k OH

GdL + OH− HooooI Gd(L)H−1 + H 2O

(4)

k −OH

where kOH and k−OH are the rate constants of the forward and backward reactions, respectively. The corresponding reaction rates are vOH = kOH[GdL][OH−] ; v−OH = k−OH[Gd(L)H−1]. The exchange lifetime of the alcoholic −OH proton is τpr = (kOH[OH−])−1. The acid and base forms, GdL and Gd(L)H−1, are expected to have different r1is and r1os contributions to the overall relaxivity. On the basis of these considerations, eq 1 can be rewritten as follows. r1p =

⎡ 1 + GdL is + os ⎢ r1 + + KH[H ] 1 + KH[H ] ⎣

+

⎤ cKH[H+] 1 ⎥ − −1 H 111.1 T1Pr + (k OH[OH ]) ⎦

GdL is+os r1

Gd(L)H‑1 is+os r1

Gd(L)H−1 is + os r1

(5)

r1is

4.6 ± 0.3 (5 ± 3) × 108 (PO43−)

two times bigger than the value obtained here for the −OH proton: TH1Pr = 4.3 μs (Table 1). This difference can readily be explained by considering the structural properties of Gd(HPDO3A). Lanthanide(III)-HP-DO3A complexes exist either in the capped square antiprism (SAP) and the capped twisted square antiprism (TSAP) geometries. The ratio of SAP and TSAP isomers is about 3:2 for Gd(HP-DO3A).30 In the dominant SAP isomer, the GdIII−OH2 and GdIII−OH bond distances are 2.51 and 2.32 Å, respectively.32 The relaxation of the protons in the vicinity of GdIII ion is governed by the dipole−dipole interaction, and the corresponding longitudinal relaxation times are dependent on the distance between the GdIII ion and proton (TH1M or TH1Pr ∼ r6Gd−H). Thus, the smaller TH1Pr value of the −OH proton is due to the shorter GdIII−OH distance. The SAP and TSAP isomers of the Gd(HP-DO3A) complex are expected to behave in an analogous manner. The two isomers were treated as a single species because their relaxation effects cannot be distinguished experimentally. The reported kinetic and relaxation parameters are the weighted averages of the corresponding parameters characteristic for the SAP and TSAP isomers. The diffusion controlled OH− assisted proton exchange of −OH (kOH = 5.2 × 109 M−1 s−1, Table 1) plays a crucial role in the proton relaxation enhancement of Gd(HP-DO3A) under alkaline conditions. The results for TH1Pr and kOH lead to the conclusion that this effect (given by the last term in eq 5) is exchange rate controlled (TH1Pr ≪ 1/(kOH[OH−])) at pH < 10.5 and relaxation controlled (TH1Pr ≫ 1/(kOH[OH−])) at pH > 10.5. The results imply that direct proton exchange between the −OH group and the bulk water has negligible contribution to the relaxivity compared to the OH− assisted path (eq 4). This is the consequence of the difference in the basicity of reactants, that is, the transfer of the −OH proton to the basic OH− is preferred over the reaction with H2O. Other Brønsted bases are also expected to accelerate the proton exchange process. Thus, the relaxivity of Gd(HP-DO3A) was studied as a function of pH in the presence of HCO3−/CO32−, H2PO4−/HPO42−/ PO43−, and HEPES buffers. As shown in Figure 3, very similar pH profiles were obtained in the presence of these buffers. The pH-dependent relaxation enhancements can readily be interpreted by considering the general base catalyzed proton exchange reactions. The acid− base equilibria of the buffers (eq 6) are characterized by the corresponding protonation constants (Table 2).

independent.1 The relaxation enhancement of Gd(HP-DO3A) in the pH range 7.5−12.5 is consistent with the deprotonation and the OH− ion catalyzed proton exchange of the −OH group,18,19 which is kinetically coupled with the paramagnetic relaxation process. The protonation constant KH of the alcoholic −OH group of Gd(HP-DO3A) (eq 3) was determined by pH-potentiometry, log KH = 11.31 ± 0.04.

KH =

4.5 ± 0.2 (2.8 ± 0.8) × 105 (HEPES−)

os

where and are the sum of and r1 for Gd(HP-DO3A) and Gd(HP-DO3A)H−1, respectively. Equation 5 predicts that the relaxivity exhibits a maximum as a function of pH at pHmax = 0.5(log KH + pKW − log(T1PH × kOH)). The experimental data were fitted to eq 5 by using a nonlinear least-squares algorithm and the calculated parameters are listed in Table 1. The sum of the r1is and r1os contributions of Gd(HP-DO3A) and Gd(HP-DO3A)H−1 complexes are 3.93 and 4.07 mM−1 s−1 at 400 MHz and 298 K in the presence of 0.15 M NaCl. In the case of the clinically used monomeric GdIII complexes, the inner- (r1is) and outer-sphere (r1os) relaxation mechanisms contribute approximately equally to the observed relaxation enhancement (r1p).1 It is assumed that the same holds true for Gd(HP-DO3A). Thus, on the basis of this assumption (GdLr1is = 2.0 mM−1 s−1) using the exchange rate of the coordinated water molecule determined by 17O NMR method (Gd(HP-DO3A): τm = 640 ns for capped square antiprism isomer, τm = 8.9 ns for capped twisted square antiprism isomer),30 the longitudinal relaxation time of the inner-sphere water protons of Gd(HP-DO3A) was calculated on the basis of the Swift-Connick equation31 to be equal to TH1M = 9.0 μs, that is, in the range of the values usually found for the protons of the coordinated water.1−3 This value is about

K iH =

[HiB] [Hi − 1B][H+]

i = 1, 2, and 3

(6)

where B stands for the basic form of the given buffer in the pH 9.0−11.0 region, that is, B = HEPES−, CO32−, and PO43−. The general base-catalyzed proton exchange process is given by eq 7. k1

GdL + B HooI Gd(L)H−1 + HB+ k −1

C

(7) DOI: 10.1021/acs.inorgchem.8b00521 Inorg. Chem. XXXX, XXX, XXX−XXX

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Figure 4. Contributions of the HEPES−, CO32−, PO43−, and OH− assisted proton exchange of the −OH proton to the relaxivity of Gd(HP-DO3A). Calculations have been performed by using eq 8 ([CO32−]t = 25 mM, [PO43−]t = 50 mM, [HEPES]t = 50 mM, 400 MHz, 0.15 M NaCl, 298 K).

Figure 3. Relaxivity of Gd(HP-DO3A) as a function of pH in the absence (black diamonds) and presence of HCO3−/CO32− (blue squares), H2PO4−/HPO42−/PO43− (red triangles) and HEPES (green circles). Symbols and solid lines represent experimental and calculated relaxivity values, respectively. Calculations have been performed by using eq 8 ([CO32−]t = 25 mM, [PO43−]t = 50 mM, [HEPES]t = 50 mM, 400 MHz, 0.15 M NaCl, 298 K).

processes between conjugate acid−base pairs which are characterized by the corresponding equilibrium constant (eq 9).

Table 2. Protonation Constants (log KiH) of HEPES, CO32−, PO43−, and OH− Ions (0.15 M NaCl, 298 K) log log log a

K1H K2H K3H

HEPES−

PO43−

CO32−

OH−

7.30(2) 2.94(2)

11.72(1) 6.65(1) 1.78(1)

9.80(1) 6.00a

15.59(1)

K=

ΔpK = log KH − log K i H

According to the general proton transfer theory, the reaction takes place via a hydrogen bonded adduct between the Gd(HP-DO3A) and B. The steady state approach for the adduct yields eq 10 for k, which is the proton exchange rate constant.34

where k1 and k−1 are the rate constants of the forward and backward reactions, respectively. The corresponding reaction rates are v1 = k1[GdL][B]; v−1 = k−1[Gd(L)H−1][HB+], the exchange lifetime of the −OH proton is τpr = (kOH[OH−] + k1[B])−1 in the presence of buffers. Thus, eq 5 transforms into eq 8.

+

⎡ 1 ⎢KH[H+]GdL r1is + os + 1 + KH[H+] ⎣

(9) 34

Ref 33 (0.5 M NaCl, 298 K).

r1p =

k1 KH [GdL][B] = + = [Gd(L)H−1][HB ] k −1 K iH

k = k 0(1 + 10 pKdonor − pKacceptor)−1

where k = k1 when Gd(HP-DO3A) is the donor and k = k−1 when Gd(HP-DO3A)H−1 is the acceptor, while k0 is the diffusion controlled rate constant.34 According to eq 10, the rate of the proton transfer (k) increases with the decrease of ΔpK. In the case of ΔpK = 0, k is half of k0 because the probability of the conversion of the adduct is equal (50%) toward both directions, that is, k1 = k−1. When ΔpK ≤ −1 the proton transfer rate constant reaches its limiting value, that is, k = k0. The proton exchange rate constants of Gd(HP-DO3A) with different buffers are plotted as a function of ΔpK in Figure 5. The solid line was calculated on the basis of eq 10 assuming that the OH− assisted exchange takes place with the maximum rate, that is, k0 = kOH. The results confirm that the proton exchange reactions of the −OH group of Gd(HP-DO3A) are in line with the general proton transfer mechanism. It should be noted that the buffer is involved in very fast proton exchange

Gd(L)H−1 is + os r1

⎤ cKH[H+] 1 ⎥ H 111.1 T1Pr + (k OH[OH−] + k1[B])−1 ⎦

(10)

(8)

The experimental data were fitted to eq 8 using a nonlinear least-squares algorithm. The equilibrium concentrations of the acid and basic forms of the buffers were calculated by using the corresponding protonation constants determined by pHpotentiometry (Table 2). The rate constant kOH was fixed at the value obtained in the absence of buffers. The fitted parameters are listed in Table 1. The calculated values of TH1Pr agree outstandingly well in the presence of different buffers. In agreement with common sense expectations, this proves that the relaxation time of the −OH proton in Gd(HP-DO3A) is not affected by the presence of buffers. Contributions to the relaxivity in the presence of various buffers are shown in Figure 4. The OH− and the buffer assisted proton exchange reactions occur simultaneously, but the OH− path is dominant under alkaline conditions. Up to pH 9.0, the relaxivity is exchange controlled and the carbonate buffer has the largest contribution to the kinetic process (Figures 3 and 4). The overall proton exchange rate increases by increasing the pH and eventually the relaxivity becomes relaxation controlled. In accordance with the general kinetic model for proton exchange processes between conjugate acid−base pairs, k1 correlates very well with the basicity of the buffers and increases in the following order HEPES− < CO32− < PO43− < OH− (Table 2). This result is in line with the general kinetic model for proton exchange

Figure 5. log k1 values as a function of ΔpK for the HEPES− (green circle), CO32− (blue square), PO43− (red triangle), and OH− (black diamond) assisted exchange processes of −OH proton in Gd(HPDO3A). The solid line represents the theoretical log k1. Calculations have been performed using eq 10. D

DOI: 10.1021/acs.inorgchem.8b00521 Inorg. Chem. XXXX, XXX, XXX−XXX

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Inorganic Chemistry with the solvent and the relaxation effect of the paramagnetic center is readily transferred into the bulk water. The results indicate that the proton exchange of the coordinated ligand may provide a useful path to design a new class of efficient MRI contrast agents. The results strongly suggest that the pH-dependence of the proton exchange induced enhancement of the relaxivity can be controlled by altering the basicity of the exchanging donor group. In order to prove this concept, the relaxometric properties of Gd(HPDO3A) derivative containing the −CF3 moiety instead of the methyl group (Scheme 1) were tested. The replacement of the electron donor −CH3 with the electron withdrawing − CF3 group is expected to make the alcoholic −OH group more acidic. In agreement with this expectation, the corresponding protonation constant of Gd(CF3-HP-DO3A) (eq 3) was found to be log KH = 6.90 ± 0.04. In order to evaluate the effect of different bases for the proton exchange of the −OH proton, the relaxivity of Gd(CF3-HP-DO3A) was measured as a function of pH in the absence and in the presence of HCO3−/CO32−, H2PO4−/HPO42−/PO43−, and HEPES− buffers (Figure 6).

Figure 7. Contribution of the HEPES−, HCO3−, and HPO42− assisted proton exchange of the −OH proton to the relaxivity of Gd(CF3-HPDO3A). Calculations have been performed by the use of eq 8 ([CO32−]t = 45 mM, [PO43−]t = 45 mM (HPO42−)a, 1.0 mM (HPO42−)b, [HEPES]t = 45 mM, 400 MHz, 0.15 M NaCl, 298 K).

of Gd(CF3-HP-DO3A) and Gd(HP-DO3A) are very similar and independent from the presence of buffers. This is an indication that the structures of the two complexes are analogous with very similar GdIII − O distances for the coordinated alcoholic −OH group. On the basis of the differences in the protonation constants of the GdIII complex and the buffers, the general proton transfer kinetic model predicts considerably larger rate constants than the experimentally observed ones in the case of HCO3− and HEPES−. Furthermore, the general model predicts strict correlation between the buffer assisted proton exchange rate constants of the −OH proton (k1) and the differences in the basicities of the GdIII complex and the buffers. In contrast, k1 increases in the following order: HCO32− < HEPES− < HPO42−. Such discrepancies were not observed in the case of Gd(HP-DO3A). In the literature, significant deviations from the general proton exchange mechanism were interpreted in terms of rate determining structural rearrangements of the reactants prior to the actual proton transfer.34 While a straightforward explanation is not readily available to interpret the deviations in the systems discussed here, structural rearrangement of the complex is unlikely involved in the overall process. The proton transfer requires the formation of a hydrogen bonded adduct between the −OH group of the complex and the base from of the buffer. Thus, it is reasonable to assume that the proton transfer may be hindered by the −CF3 group, which shields the alcoholic −OH and makes less likely the formation of the hydrogen bond. The geometries of HCO3− and HEPES− appear only suitable for the formation of a hydrogen bond between the hydrogen atom of the −OH group and the basic moiety of the buffer. The tetrahedral geometry of HPO42− offers another path for the formation of the hydrogen bonded complex. The formation of a hydrogen bond between the oxygen atom of the −OH group in the complex and the hydrogen atom of HPO42− may assist the formation of the second hydrogen bond between the reactants. In fact, a double hydrogen bonded intermediate can be envisioned with a six-membered ring as it is shown in Scheme 2. The stabilization of the intermediate may result in faster proton transfer reaction. An alternative explanation for the highest effect of HPO42− on the relaxivity of Gd(CF3-HPDO3A) complex may rely on the formation of a hydrogen bond network involving the −OH group, HPO42−, and the coordinated water, which could reduce the intramolecular rotation of the inner-sphere water molecule and increase the relaxivity as it is reported recently by Caravan et al.35 The results obtained with Gd(CF3-HP-DO3A) clearly confirm that proton exchange reactions in the presence of

Figure 6. Relaxivity of the Gd(CF3-HP-DO3A) as a function of pH in the absence (black diamonds) and presence of HCO3−/CO32− (blue squares), H2PO4−/HPO42−/PO43− (red triangles, red stars), and HEPES (green circles). Symbols and solid lines represent experimental and calculated relaxivity values, respectively. Calculations have been performed by the use of eq 8 ([CO32−]t = 45 mM, [PO43−]t = 45 mM (red triangles) and 1.0 mM (red stars), [HEPES]t = 45 mM, 400 MHz, 0.15 M NaCl, 298 K).

In water, the relaxivity increases from pH 5.0 to 8.0, then reaching a plateau and remaining constant up to pH 12.0. The relaxivity increases to 4.75 mM−1 s−1 due to the deprotonation of the −OH group. This finding reflects solely the change in r1is+os because the concentration of OH− is small in the neutral pH region and the OH− assisted proton exchange of the −OH proton does not contribute to the overall relaxation enhancement. The relaxivity of Gd(CF3-HP-DO3A) shows specific pH dependence in the presence of buffers. As shown in Figure 6, H2PO4−/HPO42−/PO43− and HEPES− buffers yield significant increase of the relaxivity and even HCO3−/CO32− has a slight enhancing effect in the pH range 5.0−8.0. Again, these relaxation effects are due to proton exchange of the coordinated −OH group (eq 7). In this case, the following forms of the buffers act as a base: HEPES−, HCO3−, and HPO42−. The corresponding proton exchange rate constants (k1) and the longitudinal relaxation time of −OH proton (TH1P) were calculated by fitting the data to eq 8. (In this case, τpr = (k1[B])−1). The contributions of various exchange processes to the relaxivity of Gd(CF3-HP-DO3A) are reported in Figure 7. The GdLr1is+os and Gd(L)H‑1r1is+os values of Gd(CF3-HP-DO3A) are somewhat higher than those of the corresponding Gd(HPDO3A) complexes (Tables 1 and 3). However, the TH1Pr values E

DOI: 10.1021/acs.inorgchem.8b00521 Inorg. Chem. XXXX, XXX, XXX−XXX

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Table 3. Relaxation Parameters for the Proton Exchange Reactions of Gd(CF3-HP-DO3A) (400 MHz, 0.15 M NaCl, 298 K) GdL is+os r1 (mM−1 s−1) Gd(L)H‑1 is+os r1 (mM−1 s−1) H 6 T 1Pr × 10 (s) k1 (M−1 s−1)

4.25 ± 0.02 4.73 ± 0.01 −(OH−)

5±1 (5 ± 1) × 105 (HCO3−)

4.7 ± 0.2 (5 ± 1) × 106 (HEPES−)

5.0 ± 0.5 (3.2 ± 0.3) × 108 (HPO42−)

As shown in Figure 8, the maximum contribution to the relaxivity gradually shifts toward the less acidic region by

Scheme 2. Double Hydrogen Bonded Intermediate Formed between Gd(CF3-HP-DO3A) Complex and HPO42− Ion

various buffers may contribute substantially to the relaxation enhancement of a GdIII-complex at physiological pH. Our findings indicate that HPO42− is the most effective catalyst of the proton exchange of the Gd(CF3-HP-DO3A) −OH proton under the applied conditions. In general, the results strongly suggest that other buffers with appropriate features may significantly enhance the relaxivity of GdIII complexes possessed by coordinated donor groups with exchangeable protons. It is evident that such a process can be utilized for diagnostic purposes in biological systems only if the maximum relaxivity enhancement occurs in the neutral/slightly acidic pH range. In human bodies, the extracellular fluid contains about 1−2 mM inorganic phosphate,36 but the phosphate buffer has a limited acceleration effect on the proton exchange process at this concentration level (Figures 6 and 7). However, the blood plasma and the interstitial fluid contain several other conjugate acid−base pairs (e.g., carbonate, organic acids, amino acids, protein side chains, etc.) with log KH values around 7.34 The concentrations of these species exceed that of the inorganic phosphate and may be important catalysts of the proton exchange process under physiological conditions. In fact, the relaxivity of Gd(CF3-HP-DO3A) measured at pH = 7.4 and 8.0 in Seronorm was found to be 5.36 and 5.00 mM−1 s−1 (400 MHz and 298 K), which confirms the contribution of all endogenous buffers to the base catalyzed proton exchange process of the Gd(CF3-HP-DO3A) −OH proton at physiological condition. The role of carbonate ion is of particular interest because this species is present at relatively large in vivo concentration ([CO32−]t = 25 mM).36 However, even at such concentration level, the proton exchange reaction of Gd(HP-DO3A) with carbonate ion does not yield any detectable contribution to the relaxivity in the neutral pH range (Figures 3 and 4). As demonstrated in this paper, the relaxivity of Gd(HP-DO3A) type complexes can be affected by changing various parameters. However, the pH and the total carbonate content cannot be varied as far as in vivo applications are concerned. If the proton exchange reaction complies with the general proton transfer model (eq 10), the only parameter for tuning the pH dependence of the relaxivity is the protonation constant of the −OH group (KH). This possibility was tested by model calculations. The relaxivity as a function of pH was calculated in the presence of 25 mM carbonate ion by varying log KH between 6−13. The proton exchange rate constants (k) were calculated using k0 = 5.2 × 109 M−1 s−1 in eq 10).

Figure 8. Model calculations: Relaxation enhancement (r1pr) caused by the CO32− and OH− assisted proton exchange of the coordinated −OH proton. Calculations of r1pr values were performed on the basis of eq 2 (log KH = 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0, and 13.0; [CO32−]t = 25 mM, TH1Pr = 4.5 μs).

increasing KH. In order to reach maximum relaxivity at pH = 7.4 the log KH value needs to be about 9.0. The calculations also indicate that the relaxation enhancement due to the −OH proton exchange can be as high as r1pr = 1.8 mM−1 s−1, which is comparable with the inner-sphere contribution of the Gd(HPDO3A) and other clinically used Gd3+ based MRI contrast agents. These findings open new possibilities for designing GBCAs with improved relaxivity features.

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CONCLUSIONS The results reported here indicate that a ligand with exchangeable proton(s) may have a significant contribution to the relaxivity of GBCAs. This work also contributes to a better understanding of the main factors affecting the proton exchange processes of the coordinated hydroxyl group. When the coordinated hydroxyl functionality is the source of the proton exchange contribution, TH1Pr is about 50% shorter than the corresponding relaxation time for the protons of the coordinated water molecule. This is due to the shorter GdIII− H distance. The relaxivity of Gd(HP-DO3A) increases to about 5.5 at pH 10.5 in the presence of various buffers due to the exchanging −OH group. However, this enhancement cannot be utilized for in vivo diagnosis for obvious reasons. The results with the −CF3 derivative clearly demonstrate that the same enhancement can be shifted toward lower pH regions by altering the pKa of the coordinating OH group. These novel findings may pave the way to design new GBCAs in which the ligand around the central GdIII ion brings functionalities with labile proton(s) even closer to the paramagnetic center. We believe that such basic research keeps providing new ideas to the quest for novel CAs with even higher relaxivities. Ultimately, a new generation of GBCAs can be produced with enhanced relaxometric properties that will allow to tackle the challenges of reduced CA doses without compromising their clinical performance. F

DOI: 10.1021/acs.inorgchem.8b00521 Inorg. Chem. XXXX, XXX, XXX−XXX

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AUTHOR INFORMATION

Corresponding Author

*Tel.: 040-3757842. Fax: 040-3757831. E-mail: zsolt.baranyai@ bracco.com. ORCID

István Fábián: 0000-0002-4467-2912 Zsolt Baranyai: 0000-0001-6844-7974 Funding

Financial support from Regione Piemonte (Gadoplus Project) is kindly acknowledged. Support from the Hungarian Research Fund (OTKA) under Grant No. K 124983 is appreciated. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The project has been carried out in the frame of the EU COST Action CA15209: European Network on NMR Relaxometry. REFERENCES

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DOI: 10.1021/acs.inorgchem.8b00521 Inorg. Chem. XXXX, XXX, XXX−XXX